Positive Inotropic Response Research Articles

Differential regulation of β2‐adrenoceptor‐mediated inotropic and lusitropic response by PDE3 and PDE4 in failing and non‐failing rat cardiac ventricle

β-Adrenoceptors play a major role in regulating myocardial function through cAMP-dependent pathways. Different phosphodiesterases (PDEs) regulate intracellular cAMP-pools and thereby contribute to the compartmentalization of cAMP-dependent effects. We explored the involvement of PDEs in limiting the β(2) adrenoceptor-mediated positive inotropic (PIR) and lusitropic (LR) responses in sham-operated (Sham) and failing rat hearts. Extensive myocardial infarctions were induced by coronary artery ligation in Wistar rats. Rats developing heart failure were studied 6 weeks after surgery. Contractility was measured in left ventricular strips from failing and Sham hearts. cAMP was quantified by RIA. In ventricular strips, stimulation of β(2) -adrenoceptors with (-)-adrenaline (300 nM CGP20712A present) exerted a small PIR and LR. In Sham hearts, β(2) -adrenoceptor-mediated as well as β(1) -adrenoceptor-mediated PIR and LR were increased by selective inhibition of either PDE3 (1 µM cilostamide) or PDE4 (10 µM rolipram). In failing rat hearts, PDE3 inhibition enhanced PIR and LR to both β(1) - and β(2) -adrenoceptor stimulation while PDE4 inhibition had no effect on these responses despite a significant increase in cAMP levels. Combined PDE3/4 inhibition further enhanced the PIR and LR of β(2) - and β(1) -adrenoceptor activation both in Sham and failing hearts, compared with PDE3 inhibition alone. PDE4 enzyme activity was reduced in failing hearts. Both PDE3 and PDE4 attenuated β(2) - and β(1) -adrenoceptor-mediated contractile responses in Sham hearts. In failing hearts, these responses are attenuated solely by PDE3 and thus even selective PDE3 inhibitors may provide a profound enhancement of β-adrenoceptor-mediated responses in heart failure.

Differential effects of adenosine A2aand A2breceptors on cardiac contractility

The mammalian myocardium expresses four adenosine receptor (AR) subtypes: A(1)AR, A(2a)AR, A(2b)AR, and A(3)AR. The A(1)AR is well known for its profound antiadrenergic effects, but the roles of other AR subtypes in modulating contractility remain inconclusive. Thus, the objective of this study was to determine the direct and indirect effects of A(2a)AR and A(2b)AR on cardiac contractility. Experiments were conducted in paced, constant pressure-perfused isolated hearts from wild-type (WT), A(2a)AR knockout (KO), and A(2b)AR KO mice. The A(2a)AR agonist CGS-21680 did not alter basal contractility or β-adrenergic receptor agonist isoproterenol (Iso)-mediated positive inotropic responses, and Iso-induced effects were unaltered in A(2a)AR KO hearts. However, A(2a)AR gene ablation resulted in a potentiation of the antiadrenergic effects mediated by the A(1)AR agonist 2-chloro-N-cyclopentyladenosine. The nonselective AR agonist 5'-N-ethylcarboxamido adenosine and the selective A(2b)AR agonist BAY 60-6583 induced coronary flow-independent increases in contractility, but BAY 60-6583 did not alter Iso-induced contractile responses. The A(1)AR antiadrenergic effect was not potentiated in A(2b)AR KO hearts. The expression of all four AR subtypes in the heart and ventricular myocytes was confirmed using real-time quantitative PCR. Taken together, these results indicate that A(2a)AR does not increase cardiac contractility directly but indirectly alters contractility by modulating the A(1)AR antiadrenergic effect, whereas A(2b)AR exerts direct contractile effects but does not alter β-adrenergic or A(1)AR antiadrenergic effects. These results indicate that multiple ARs differentially modulate cardiac function.

How does CaMKIIδ phosphorylation of the cardiac ryanodine receptor contribute to inotropy?

In PNAS, Kushnir et al. (1) reported that knocking out the CaMKII S2814 phosphorylation site on the ryanodine receptor (RyR) decreases the positive inotropic response to an increase of stimulation frequency. In previous work, this group has reported that phosphorylation by CaMKII increases the open probability of the RyR (2). They now suggest that the RyR-CaMKII effect contributes to frequency-dependent inotropy in normal mice. Furthermore, they contend that impaired phosphorylation of the RyR contributes to the decreased inotropic effect of increasing frequency in heart failure. We think it is essential to comment on these conclusions.

Effects of GH Secretagogues on Contractility and Ca2+Homeostasis of Isolated Adult Rat Ventricular Myocytes

Ghrelin and its synthetic analogue hexarelin are specific ligands of GH secretagogue receptor (GHS-R) and induce a variety of cardiovascular protective and cardiac positive inotropic effects. The signaling system underlying immediate effects of both GHSs in cardiomyocytes remains undefined. In the present study, we investigated the immediate effects of GHSs on isolated ventricular myocyte shortening, intracellular Ca(2+) ([Ca(2+)](i)) transients, and the L-type Ca(2+) current (I(Ca,L)). Putative intracellular signalling cascades were studied with specific receptor and signalling blockers. In fresh isolated adult Wistar rat ventricular myocytes, GHSs produced a positive inotropic effect in a concentration-dependent manner and increased the amplitude of [Ca(2+)](i) transients and the I(Ca,L). The positive inotropic response was abolished by the GHS-R1a antagonist [D-Lys(3)]-GH-releasing peptide-6 (10 microm). GHS-induced increase in the I(Ca,L) was abolished by [D-Lys(3)]-GH-releasing peptide-6 and protein kinase C inhibitor, chelerythrine chloride (5 microm), but not by protein kinase A inhibitor, KT 5720 (10 microm). We conclude that hexarelin and ghrelin increase the I(Ca,L), through GHS-R1a receptor and protein kinase C signalling cascade, which contribute to its direct positive inotropic effect on cardiomyocytes.

Overexpression of β 2AR improves contractile function and cellular survival in rabbit cardiomyocytes under chronic hypoxia

Chronic hypoxia usually evokes sustained release of endogenous neurohormones, leading to β 2-adrenergic receptor (β 2AR) desensitization and downregulation of expression, which impacts cellular contractility. We investigated whether exogenous β 2AR could compensate for the functional deficiency of β 2AR in rabbit cardiomyocytes under chronic hypoxia, and whether this led to improved contractility and cellular survival. A surgical experimental model of cyanotic heart disease was established in rabbits. Adv.hβ 2AR was transfected into cardiomyocytes isolated from animals subjected to 6-week systemic hypoxia. The levels of cellular contractile function, protein expression of hβ 2AR, p-Akt, p-Erk, and caspase-3, and cellular survival pre- and post-Adv.hβ 2AR delivery were determined. In the cyanotic cells, decreased shortening and lengthening of TPC and R50 were evident. Cellular diastolic functioning showed greater deterioration compared to the systolic function ( P < 0.05). In cyanotic cells, the positive inotropic response to isoproterenol was decreased ( P < 0.01), low levels of cellular survival were found, protein levels of β 2AR, p-Akt, and p-Erk were downregulated, and protein levels of caspase-3 were upregulated. After Adv.hβ 2AR delivery, enhanced contractile function was achieved ( P < 0.01), TPC and R50 levels recovered up to 99% and 81.7% of the normal control levels, respectively ( P < 0.05), and cellular survival improved ( P < 0.01). Our results demonstrate that overexpression of the β 2AR gene in cardiomyocytes exposed to chronic hypoxia provides significant catecholamine-dependent inotropic support and cellular protection.

Effects of long-term pretreatment with isoproterenol on inotropic responsiveness to alpha-adrenoceptor stimulation: study in isolated perfused rat hearts.

The effects of chronic pretreatment with isoproterenol (5 mg kg(-1)) daily for 10 days on cardiac alpha-adrenergic responsiveness in Langendorff heart preparations were investigated. Isoproterenol pretreatment caused cardiac hypertrophy (29%) as shown by a significant increase in the ratio of ventricular dry weight to body weight. In preparations from isoproterenol-pretreated rats, both maximum increases in left ventricular systolic pressure and heart rate elicited by isoproterenol (10(-12) to 10(-4) M) were significantly reduced (the isoproterenol concentration producing 50% of the maximum positive inotropic and chronotropic responses was enhanced almost 32- and 4-fold, respectively), while the positive inotropic response to phenylephrine (10(-12) to 10(-4) M) was significantly enhanced (the phenylephrine concentration producing 50% of the maximum positive inotropic effect was reduced almost 100-fold), compared with saline-pretreated rats. In preparations from both groups, phenylephrine infusion induced non-significant changes in heart rate and its positive inotropic response was reduced in the presence of propranolol (10(-7) M) in the perfusion medium. Even under beta-adrenoceptor blockade, the curve for the phenylephrine-induced positive inotropic effect remained shifted upward after isoproterenol pretreatment. Chronic isoproterenol pretreatment induces the expected cardiac beta-adrenoceptor desensitization while simultaneously enhancing the positive inotropic responsiveness to phenylephrine in Langendorff heart preparations. These findings support the hypothesis that cardiac alpha1-adrenoceptor stimulation may contribute to the maintenance of myocardial function under conditions in which beta-adrenoceptor function is compromised.

Alpha-adrenergic responsiveness in rat isolated perfused heart after abdominal aortic coarctation.

Chronic isoproterenol pre-treatment, a well-known model of compensatory hypertrophy associated with cardiac beta-adrenoceptor desensitization, enhances the inotropic response to phenylephrine in rat isolated perfused hearts, supporting the hypothesis that myocardial alpha-adrenoceptor stimulation contributes to the maintenance of myocardial performance in situations in which cardiac beta-adrenoceptor function is compromised. To further corroborate this hypothesis, the effects of abdominal aortic coarctation on cardiac alpha-adrenergic responsiveness were investigated in Langendorff heart preparations. Abdominal aortic coarctation causes cardiac hypertrophy (21%) as shown by a significant increase in the ratio of ventricular dry weight to bodyweight. In preparations from hypertrophied rats, both maximum increases in left ventricular systolic pressure and heart rate elicited by isoproterenol (10(-12) to 10(-4) M) were significantly reduced (the isoproterenol concentration producing 50% of the maximum positive inotropic and chronotropic responses was enhanced almost 21- and 2-fold, respectively). However, the positive inotropic response to phenylephrine (10(-12) to 10(-4) M) remained unaffected following abdominal aortic coarctation, when compared with sham-operated rats. In preparations from both groups, phenylephrine infusion did not induce significant changes in heart rate. These results show that although abdominal aortic stenosis induced desensitization of cardiac beta-adrenoceptors, it did not enhance cardiac alpha-adrenoceptor responsiveness. This suggests that such an enhancement depends on the experimental model used to induce cardiac hypertrophy associated with desensitization of cardiac beta-adrenoceptors.

The effects of streptozotocin-induced diabetes and aldose reductase inhibition with sorbinil, on left and right atrial function in the rat.

Diabetes mellitus is frequently associated with the complications of cardiovascular disease. Activation of the aldose reductase (or polyol) pathway has long been implicated as an underlying factor for the development of many diabetic complications and indeed, treatment with aldose reductase inhibitors has been shown to prevent or reverse many of these diabetic complications. This study examines the effects of 14-day streptozotocin-induced diabetes on alpha1- and beta-adrenoceptor-mediated responses in rat isolated left and right atria. The effects of treatment with the aldose reductase inhibitor (ARI) sorbinil were also studied. A positive inotropic response was observed to both isoprenaline and phenylephrine in left atria. Diabetes of 14 days duration resulted in a supersensitivity of these tissues to the beta-adrenoceptor agonist in comparison with controls, while responses to the alpha1-adrenoceptor agonist were unaltered. Spontaneously beating right atria from diabetic rats was found to have a depressed resting rate compared with control tissues, although positive chronotropic beta-adrenoceptor-mediated responses were not affected by diabetes. Phenylephrine produced alpha1-adrenoceptor-mediated chronotropic responses in right atrial tissues, and these were found to be enhanced in rats with diabetes. Treatment of diabetic rats with the ARI sorbinil was successful in preventing only one of the observed diabetes-induced changes in atrial function, namely the supersensitivity of left atria to isoprenaline. Sorbinil treatment did, however, alter responses of control left and right atria in a manner similar to diabetes. In conclusion, streptozotocin-induced diabetes of 14 days duration was found to cause a number of alterations in the functioning of both left and right atria. ARI treatment with sorbinil failed to prevent all but one of these changes, and in addition altered responses of atria from control rats, having a similar effect to that produced by diabetes. These data suggest that sorbinil may have effects in addition to, and independent of, aldose reductase inhibition in the cardiovascular system.

Cod liver oil supplementation improves cardiovascular and metabolic abnormalities in streptozotocin diabetic rats

Abnormalities in the metabolism of essential fatty acids and the results of increased oxidative stress have been implicated in cardiovascular disorders observed in diabetes mellitus. This study, therefore, aimed to investigate the effects of cod liver oil (CLO, Lysi Ltd, Iceland), which comprises mainly an antioxidant vitamin A, n:3 polyunsaturated fatty acids (n:3 PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on cardiovascular abnormalities in streptozotocin (STZ)-diabetic rats. Two days after single STZ (55 mg kg(-1), i.p.) or vehicle injection, diabetes was verified by increased blood glucose, and non-diabetic and diabetic rats were left untreated or treated with CLO (0.5 mL kg(-1) daily, by intragastric probing) for 12 weeks. Plasma glucose, triacylglycerol and cholesterol concentrations were significantly elevated in 12-week untreated-diabetic rats; CLO provided better weight gain, entirely prevented the plasma lipid abnormalities, but partially controlled the glycaemia in diabetic rats. In isolated aorta rings, diabetes resulted in increased phenylephrine-induced vasoconstriction and isoprenaline-induced vasorelaxation, impaired endothelium-dependent vasodilatation and unchanged responsiveness to sodium nitroprusside. CLO treatment completely prevented endothelial deficiency, partly corrected the phenylephrine-induced vasoconstriction and did not affect the responses to isoprenaline and sodium nitroprusside in diabetic aorta. Diabetes also produced a marked decrease in the rate of spontaneously beating right atria and a significant increase in basal contractile force of left ventricular papillary muscle. The responsiveness of right atria to the positive chronotropic effect of isoprenaline was significantly decreased in diabetic rats, and was increased in CLO-treated diabetic rats. The positive chronotropic effect of noradrenaline was markedly increased in diabetic atria, but prevented by CLO treatment. Diabetes also resulted in an increased positive inotropic response of papillary muscle to both noradrenaline and isoprenaline, which were prevented by CLO treatment. CLO treatment also resulted in lower tissue sensitivity (pD(2)) to these agonists in diabetic papillary muscle. Ventricular hydroxyproline content was found to be unchanged among the experimental groups. The ultrastructure of diabetic myocardium displayed various degenerations (i.e. intracellular oedema, myofibrillar fragmentation, condensed pleomorphic mitochondria, thick capillary irregular basement membrane, swollen endothelial cells), which were partially prevented by CLO treatment. We conclude that the supplementation with CLO is effective in preventing cardiovascular disorders observed in experimental diabetes.

Pharmacological investigation on nigrescigenin-a cardenolide from Parquetina nigrescens (Afzel.) Bullock: comparative studies on cardiotonic effects of Parquetina nigrescens, g-strophanthin and noradrenaline in guinea-pig isolated atria.

The cardiotonic and catecholamine-like effects of Parquetina nigrescens extract-induced contractile force of guinea-pig left and right atria were investigated in-vitro. Isometric contractions were recorded. P. nigrescens extract, 5-150 microg mL(-1), increased the force of contraction dose dependently in electrically driven left atria. The concentration of P. nigrescens extract producing 50% of the maximal effect (EC50 value) was 7.5 microg mL(-1). The positive inotropic response differed from that of g-strophanthin by its high rate of onset and its complete reversibility upon removal of the extract from the incubation medium. In spontaneously beating right atrial muscle, P. nigrescens extract increased the rate of contractions. Its positive chronotropic and inotropic effects were partly antagonized by propranolol and atenolol indicating the presence of an adrenergic acting principle in P. nigrescens extract. In contrast, the inotropic response to P. nigrescens extract could not be completely suppressed by beta-blocking agents, suggesting that the force of contraction is not only increased by a sympathomimetic ingredient of P. nigrescens extract but also by the cardenolides known to be present in P. nigrescens.

Effects of mild hypothermia on hemodynamics in cardiac arrest survivors and isolated failing human myocardium

Post-cardiac arrest myocardial dysfunction is a common phenomenon after return of spontaneous circulation (ROSC) and contributes to hemodynamic instability and low survival rates after cardiac arrest. Mild hypothermia for 24 h after ROSC has been shown to significantly improve neurologic recovery and survival rates. In the present study we investigate the influence of therapeutic hypothermia on hemodynamic parameters in resuscitated patients and on contractility in failing human myocardium. We analyzed hemodynamic data from 200 cardiac arrest survivors during the hypothermia period. The initial LVEF was 32.6 ± 1.2% indicating a significantly impaired LV function. During hypothermia induction, the infusion rate of epinephrine could be significantly reduced from 9.1 ± 1.3 μg/min [arrival intensive care unit (ICU) 35.4°C] to 4.6 ± 1.0 μg/min (34°C) and 2.8 ± 0.5 μg/min (33°C). The dobutamine and norepinephrine application rates were not changed significantly. The mean arterial blood pressure remained stable. The mean heart rate significantly decreased from 91.8 ± 1.7 bpm (arrival ICU) to 77.3 ± 1.5 bpm (34°C) and 70.3 ± 1.4 bpm (33°C). In vitro we investigated the effect of hypothermia on isolated ventricular muscle strips from explanted failing human hearts. With decreasing temperature, the contractility increased to a maximum of 168 ± 23% at 27°C (n = 16, P < 0.05). Positive inotropic response to hypothermia was accompanied by moderately increased rapid cooling contractures as a measure of sarcoplasmic reticulum (SR) Ca2+ content, but can be elicited even when the SR Ca2+ release is blocked in the presence of ryanodine. Contraction and relaxation kinetics are prolonged with hypothermia, indicating increased Ca2+ sensitivity as the main mechanism responsible for inotropy. In conclusion, mild hypothermia stabilizes hemodynamics in cardiac arrest survivors which might contribute to improved survival rates in these patients. Mechanistically, we demonstrate that hypothermia improves contractility in failing human myocardium most likely by increasing Ca2+-sensitivity.Electronic supplementary materialThe online version of this article (doi:10.1007/s00392-010-0113-2) contains supplementary material, which is available to authorized users.

Differential effects of lumbar and thoracic epidural anaesthesia on the haemodynamic response to acute right ventricular pressure overload

The safety of epidural anaesthesia in patients at risk for right ventricular pressure overload remains controversial. We compared the haemodynamic effects of vascular and cardiac autonomic nerve block, induced by selective lumbar (LEA) and high thoracic epidural anaesthesia (TEA), respectively, in an animal model subjected to controlled acute right ventricular pressure overload. Eighteen pigs were instrumented with epidural catheters at the thoracic (T) and lumbar (L) level and received separate injections at T2 (1 ml) and L3 (4 ml) with saline (s) or bupivacaine 0.5% (b). Three groups of six animals were studied: (i) a control group (Ls+Ts), (ii) LEA group (Lb+Ts), and (iii) TEA group (Ls+Tb). Haemodynamic measurements including biventricular pressure-volumetry were performed. Right ventricular afterload was then increased by inflating a pulmonary artery (PA) balloon. Measurements were repeated after 30 min of sustained right ventricular afterload increase. LEA decreased systemic vascular resistance (SVR) and did not affect ventricular function. TEA had minor effects on SVR but decreased left ventricular contractility while baseline right ventricular function was not affected. Control and LEA-treated animals responded similarly to a PA balloon occlusion with an increase in right ventricular contractility and heart rate. Animals pretreated with a TEA did not show this positive inotropic response and developed low cardiac output in the presence of right ventricular pressure overload. In contrast to LEA, TEA reduced the haemodynamic tolerance to PA balloon occlusion by inhibiting the right ventricular positive inotropic response to acute pressure overload.

Opposing cardiac effects of autoantibody activation of beta-adrenergic and M2 muscarinic receptors in cardiac-related diseases

Background Activating autoantibodies to β-adrenergic receptors (AAβ1/2AR) and M2 muscarinic receptors (AAM2R) have been reported in several cardiac diseases and may have pathophysiologic relevance. However, the interactions and relative effects of AAβ1AR, AAβ2AR and AAM2R on contractile function have not been characterized. Methods The inotropic effects of IgG from 18 selected patients with cardiomyopathy and/or atrial tachyarrhythmias positive by ELISA for antibodies to β1/2AR were studied using an isolated canine Purkinje fiber contractility assay. M2R-blockade was tested using atropine while selective β1AR and β2AR blockade used CGP-20712A and ICI-118551 respectively. Results Fifteen of the 18 anti-β1/2AR ELISA-positive samples demonstrated evidence for negative inotropic muscarinic effects which were blocked using atropine. Atropine failed to uncover a positive inotropic response in 2 of the 18 IgG samples (false positive ELISA for AAβAR). In the remaining 16 AAβAR true-positive subjects, the β1AR-induced increase in contractility (concurrent M2/β2 blockade) was augmented to 140.5 ± 12.2% of baseline compared to 127.4 ± 7.2% of baseline with M2 blockade (atropine) only ( p < 0.001, n = 16). The β2AR-induced increase in contractility (concurrent M2/β1 blockade) was only 114.5 ± 4.3% of baseline ( p < 0.001, n = 16). Combined M2 and β1/β2 blockade eliminated any increase in contractility. Conclusions The inherently positive inotropic effect of AAβ1AR was negatively modulated by AAM2R and AAβ2AR. These opposing effects of receptor-activating autoantibodies may alter cardiac performance and influence clinical outcome depending on their receptor type and relative contractile activity.

Cellular Mechanisms of Contractile Impairment in Human Chronic Atrial Fibrillation

Chronic Atrial Fibrillation (cAF) is associated with contractile impairment. Down regulation of L-Type Ca2+current plays a major role in determining contractile dysfunction. However, additional EC-Coupling changes could be involved in human cAF. We dissected atrial trabeculae from left atrial appendages of cAF patients undergoing cardiac surgery and used them for simultaneous force and action potential recordings. Cells isolated from the same samples were used for Ca2+current, Ca2+transients, and reticular Ca2+content(caffeine) measurements. Samples from sinus rhythm (SR) patients were used as controls. Despite 75%reduction in basal force, positive inotropic responses to isoproterenol, stimulation pauses, and high [Ca2+]e were preserved in cAF. Basal Ca2+current and Ca2+transients were decreased (30%of SR) but showed a greater increase upon inotropic stimuli, reducing the difference with SR. No difference was found in time-course of mechanical restitution, suggesting no major impairment of Ryanodine Receptor function. The finding that sarcoplasmic reticulum Ca2+ content was not reduced in cAF suggests that Ca2+release impairment could be due to a change from synchronous EC Coupling to propagated Ca2+-induced Ca2+-release (CICR), in which a fast subsarcolemmal Ca2+rise is followed by Ca2+diffusion-mediated signal propagation toward the cell core. The following observations in cAF preaparations supports this idea: 1)Ca2+transients showed a fast monophasic rise(subsarcolemmal Ca2+-release only) but exhibited a biphasic, dome-shaped aspect (peripheral rise followed by inward Ca2+-wave spread) upon inotropic stimulations; 2)Ca2+recirculation fraction decreased, suggesting an increased role of NCXvs.SERCA, consistent with a non-propagated peripheral Ca2+rise; 3)twitch force transitorily increased after abrupt reduction of intracellular Ca2+ buffering by the SERCA blocker CPA. Reduction in T-tubules density and/or increased cytosolic Ca2+buffering (e.g.increased myofilament Ca2+sensitivity) could be crucial in cAF for transition to propagated-CICR and Ca2+-wave spread impairment. Ca2+trigger enhancement or Ca2+diffusion improvement could promote recruitment of deeper myofibril layers and increase twitch force.

Heart failure switches the RV α1-adrenergic inotropic response from negative to positive

Right ventricular (RV) failure is a serious common clinical problem that is poorly understood. Therefore, for failing and nonfailing hearts, we examined the distinctive inotropic responses induced in the RV myocardium after the stimulation of alpha(1)-adrenergic receptors (ARs). In RV trabeculae from nonfailing mouse hearts, alpha(1)-ARs induced a negative inotropic response, consistent with our previous study. In marked contrast, in RV trabeculae from failing hearts, 12 wk after coronary artery ligation, alpha(1)-ARs induced a positive inotropic response. Mechanistically, experiments with skinned trabeculae showed that alpha(1)-ARs decreased myofilament Ca(2+) sensitivity in the nonfailing RV myocardium, whereas alpha(1)-ARs increased Ca(2+) sensitivity in heart failure. This suggests that a switch in the Ca(2+) sensitivity response to alpha(1)-AR stimulation explained the switch in the RV alpha(1)-AR inotropic response in heart failure. Myosin light chain kinase (MLCK) can increase myofilament Ca(2+) sensitivity, and the smooth muscle isoform (smMLCK), which is also present in cardiomyocytes, was more abundant in the RV myocardium from failing versus nonfailing hearts. Moreover, the MLCK inhibitor ML-9 prevented the switch of the RV myocardium to a positive alpha(1)-AR inotropic response in heart failure. In the left ventricular myocardium, in contrast, alpha(1)-AR inotropic responses were not different in failing versus nonfailing hearts, and smMLCK abundance was not increased in heart failure. In relation to human disease, we found that smMLCK mRNA and protein levels were increased in RVs from failing human hearts. We conclude that the RV inotropic response to alpha(1)-ARs is switched from negative to positive in heart failure, through a pathway involving increased myofilament Ca(2+) sensitivity. Since alpha(1)-AR agonist catecholamines are elevated in heart failure, increased alpha(1)-AR inotropic responses in the RV myocardium may be adaptive in heart failure by helping the failing RV respond to increased pulmonary pressures.

Cardiovascular effects of scorpionfish ( Scorpaena plumieri) venom

The aim of the present study was to investigate the cardiovascular activity of Scorpaena plumieri venom in both in vivo and in vitro models. In anesthetized rats, doses of the venom (14–216 μg protein/kg) induced a transient increase in the mean arterial pressure. However at higher dose (338 μg protein/kg) this effect was followed by a sudden hypotension and the animal evolved to death. The heart rate was temporarily increased and followed by bradycardia using doses ≥108 μg/kg. In isolated rat hearts the crude venom (5–80 μg protein) produced dose-dependent positive ventricular chronotropic, inotropic, lusitropic and coronary vasoconstriction responses. Partial purification of an active fraction (CF, cardiovascular fraction) which reproduced the cardiovascular effects induced by crude venom on isolated hearts was achieved by conventional gel filtration chromatography. Adrenergic blockades, prazosin and propranolol, significantly attenuated these responses. The coronary vasoconstriction response to CF was also attenuated by chemical endothelium denudation. In conclusion, the data showed that S. plumieri fish venom induces disorders in the cardiovascular system. It also suggests that α 1 and β-adrenergic receptors, and the vascular endothelium, are involved at least partially, in these cardiac effects.

Involvement of Cyclooxygenase-2 in Carbachol-Induced Positive Inotropic Response in Mouse Isolated Left Atrium

The mouse heart is expected to have characteristic contractile properties. However, basic information on the function of the mouse heart has not been accumulated sufficiently. In this study, the involvement of cyclooxygenase (COX)-2 in carbachol (CCh)-induced inotropic response was investigated in mouse isolated left atrium. Influences of CCh and their mechanisms of action on developed tension elicited by electrical stimulation were examined pharmacologically. The presence of COX-2 in atrium was examined by Western blotting and immunohistochemical analysis. CCh (3 microM for 15 min) produced a biphasic inotropic response: a transient decrease in contractile force followed by a late increase. Atropine suppressed the biphasic inotropic response to CCh. A muscarinic M(3) receptor antagonist, 4-diphenyl-acetoxy-N-methlpiperidine, inhibited the late positive inotropic action. Blockade of prostaglandin (PG) E(2) or F(2alpha) receptor by 6-isopropoxy-9-oxoxanthene-2-carboxylic acid (AH6809) or 9alpha, 15R-dihydroxy-11beta-fluoro-15-(2,3-dihydro-1H-inden-2-yl)-16,17,18,19,20-pentanor-prosta 5Z, 13E-dien-1-oic acid (AL8810), respectively, significantly suppressed the positive inotropic response to CCh. A nonselective COX inhibitor, indomethacin, and a selective COX-2 inhibitor, N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS-398) inhibited the positive response. A COX-1 inhibitor, valeroyl salicylate, did not affect the positive response. The positive response was almost completely abolished in the endocardial endothelium-deprived atria. Existence of COX-2 in endocardial endothelium was confirmed by Western blotting and immunohistochemical analysis. The present study indicated that the CCh-induced positive inotropic response was mediated by PGs, possibly PGE(2) and PGF(2alpha), released in part from endocardial endothelium. Furthermore, for the first time, we demonstrated that the production of PGs depended in part on COX-2 in endocardial endothelium through the muscarinic M(3) receptor stimulation.

Phosphodiesterases do not limit β1-adrenoceptor-mediated sinoatrial tachycardia: evidence with PDE3 and PDE4 in rabbits and PDE1–5 in rats

The mammalian heart expresses at least five phosphodiesterases (PDE1-5). Catecholamines produce surges of inotropically relevant cAMP through beta(1)-adrenoceptor stimulation. cAMP is mainly hydrolysed by PDE3 and/or PDE4 thereby blunting contractility. Basal sinoatrial beating rate in mouse, rat, piglet and rabbit sinoatrial cells is reduced by PDE3 and/or PDE4 through hydrolysis of cAMP. However, in rodents, the tachycardia elicited by catecholamines through production of cAMP by beta-adrenoceptor activation is not controlled by PDE3 and PDE4, despite a blunting effect of PDE3 or/and PDE4 on basal sinoatrial beating, but it is unknown whether PDE3 limits catecholamine-evoked tachycardia in the rabbit. Since rabbit sinoatrial cells are an important model for pacemaker research, we investigated whether the positive chronotropic effects of (-)-noradrenaline on spontaneously beating right atria of the rabbit are potentiated by inhibition of PDE3 with cilostamide (300 nM). We also studied the sinoatrial effects of the PDE4 inhibitor rolipram (10 microM) and its influence on the responses to (-)-noradrenaline. For comparison, we investigated the influence of cilostamide and rolipram on the positive inotropic responses to (-)-noradrenaline on rabbit left atria and right ventricular papillary muscles. Cilostamide and concurrent cilostamide + rolipram, but not rolipram alone, increased sinoatrial rate by 15% and 31% of the effect of (-)-isoprenaline (200 microM) but the PDE inhibitors did not significantly change the chronotropic potency of (-)-noradrenaline. In contrast in papillary muscle, the positive inotropic effects of (-)-noradrenaline were potentiated 2.4-, 2.6- and 44-fold by cilostamide, rolipram and concurrent cilostamide + rolipram, respectively. In left atrium, the positive inotropic effects of (-)-noradrenaline were marginally potentiated by cilostamide, as well as potentiated 2.7- and 32-fold by rolipram and by concurrent cilostamide and rolipram respectively. To compare the influence of PDE1-5 on basal sinoatrial rate and (-)-noradrenaline-evoked tachycardia, we investigated on rat right atria the effects of selective inhibitors. The PDE4 inhibitor rolipram and non-selective inhibitor isobutyl-methylxanthine caused tachycardia with -logEC(50)s of 7.2 and 5.0 and E(max) of 18% and 102% of (-)-isoprenaline, respectively. Rolipram did not change the chronotropic potency of (-)-noradrenaline. At high concentrations (10-30 microM), the PDE1, PDE3 and PDE5 inhibitors 8-methoxymethyl-3-isobutyl-1-methylxanthine, cilostamide and sildenafil, respectively, caused marginal tachycardia but did not significantly change the chronotropic potency of (-)-noradrenaline. The PDE2-selective inhibitor erythro-9-[2-hydroxy-3-nonyl]adenine caused marginal bradycardia at 30 microM and tended to reduce the chronotropic potency of (-)-noradrenaline. Rabbit PDE3 reduces basal sinoatrial rate. Although PDE4 only marginally reduces rate, under conditions of PDE3 inhibition, it further reduces sinoatrial rate. Both PDE3 and PDE4 control atrial and ventricular positive inotropic effects of (-)-noradrenaline. In contrast, neither PDE3 nor PDE4 limit the sinoatrial tachycardia induced by (-)-noradrenaline. In the rat, only PDE4, but not PDE1, PDE2, PDE3 and PDE5, reduces basal sinoatrial rate. None of the five rat PDEs limits the (-)-noradrenaline-evoked tachycardia. Taken together, these results confirm and expand evidence for our proposal that the cAMP-compartment modulating basal sinoatrial rate, controlled by PDE3 and/or PDE4, is different from the PDE-resistant cAMP compartment involved in beta(1)-adrenoceptor-mediated sinoatrial tachycardia.

Age-dependent differential crosstalk between alpha(1)-adrenergic and angiotensin receptors

Previous reports of crosstalk between alpha(1)- adrenergic receptors (alpha(1)-AR) and angiotensin receptors (ATR) have pointed to the existence of physiological regulation between the sympathetic nervous system and the renin-angiotensin system at the receptor level. This regulation may have an important role in the control of blood pressure and may be modified in different cardiovascular pathologies. Aging is considered to be an independent cardiovascular risk factor. Nevertheless, neither the variation in physiological action or interaction of signal transduction between these two receptors as a result of aging has been established. To clarify these aspects, the interaction between alpha(1)-AR and ATR was evaluated. The inotropic response of alpha(1)-AR to agonists was assessed in the presence and absence of angiotensin II using the left atria of 3.5-, 12-, 18- and 24-month-old (young adult, middle aged, elderly and aged, respectively) male Wistar rats. In the four age groups of rat hearts, the activities of tyrosine kinase were measured when just the AT(1)R subtype was activated, or when both alpha(1)-AR and AT(1)R were activated. The activities of cytosolic phospholipase A(2) and the levels of cyclic GMP were investigated when just the AT(2)R subtype was activated, or when both alpha(1)-AR and AT(2)R were activated. No effect was found on the cumulative concentration-response curve for phenylephrine when AT(1)R was activated in 3.5- or 12-month-old rats. However, in 18- and 24-month-old rats, the maximum positive inotropic response and the negative logarithm of the effective 50% concentration increased markedly. No effect was found on the cumulative concentration response curve induced by phenylephrine when AT(2)R was activated. The activities of tyrosine kinase increased significantly in 3.5- and 12-month-old rats, but there was no difference in 18- and 24-month-old rats when alpha(1)-AR and AT(1)R were both activated compared with when just AT(1)R was activated. Cytosolic phospholipase A(2) activity and cyclic GMP levels decreased significantly when both alpha(1)-AR and AT(2)R were activated compared with when just AT(2)R was activated. In the isolated left atria of elderly and aged rats, the activation of AT(1)R enhanced the positive inotropic response induced by the activation of alpha(1)-AR. The activation of AT(2)R had no effect on the positive inotropic response induced by the activation of alpha(1)-AR. The action of alpha(1)-AR increased the signal transduction of AT(1)R in young-adult and middle-aged rat hearts but had no effect in elderly and aged hearts. The action of alpha(1)-AR had no effect on AT(2)R signal transduction.

M3 Muscarinic Receptors Mediate Positive Inotropic Responses in Mouse Atria: A Study with Muscarinic Receptor Knockout Mice

The potential functional roles of M(3) muscarinic receptors in mouse atria were examined by pharmacological and molecular biological techniques, using wild-type mice, muscarinic M(2) or M(3) receptor single knockout (M(2)KO, M(3)KO), and M(2) and M(3) muscarinic receptor double knockout mice (M(2)/M(3)KO). Real-time quantitative reverse transcriptase-polymerase chain reaction analysis showed that the M(2) receptor mRNA was expressed predominantly in mouse atria but that the M(1), M(3), M(4), and M(5) receptor subtypes were also expressed at low levels. Carbachol (10 nM-30 microM) decreased the spontaneous beating frequency of right atria isolated from wild-type mice. Studies with subtype-preferring antagonists and atria from M(2)KO mice confirmed that this activity is mediated by the M(2) receptor subtype. In left atria from wild-type mice, carbachol decreased the amplitude of electrical field stimulation-evoked contractions (negative inotropic action), but this inhibition was transient and was followed by a gradual increase in contraction amplitude (positive inotropic response). In atria from M(3)KO mice, the transient negative inotropic action of carbachol changed to a sustained negative inotropic action. In contrast, in atria from M(2)KO mice, carbachol showed only positive inotropic activity. In atria from M(2)/M(3) double KO mice, carbachol was devoid of any inotropic activity. These observations, complemented by functional studies with subtype-preferring antagonists, convincingly demonstrate that atrial M(3) muscarinic receptors mediate positive inotropic effects in mouse atria. Physiologically, this activity may serve to dampen the inhibitory effects of M(2) receptor activation on atrial contractility.