Activation of immune and inflammatory systems in chronic heart failure: novel therapeutic approaches.

Despite extensive research and novel treatments, chronic heart failure (CHF) remains a cause of high morbidity and mortality. Mounting evidence suggested that immune activation and inflammation play critical roles in the pathogenesis of CHF. In this review, we examine the current evidence regarding this contemporary pathophysiological mechanism, and evaluate the effects of conventional and novel cardiovascular drugs, such as calcium sensitisers and statins, on the immune and inflammatory mediator's network. Although therapies, which specifically antagonise tumour necrosis factor-alpha have not demonstrated considerable benefit in patients with CHF, there is an increasing evidence to suggest greater value from non-specific anti-inflammatory approaches, including: pentoxifylline, intravenous immunoglobulin, immune modulation therapy, growth hormones, physical training and nutrition regulation. Several innovative therapeutic targets, such as peroxisome proliferator-activated receptor gamma activators, Rho-kinase, p38 mitogen-activated protein kinase, nuclear transcription factor NF-kappaB, recovering or augmenting parasympathetic tone, cardiac resynchronisation therapy, macrophage inhibitors and chemokine receptor antagonists, are briefly discussed in this review. While we have recently demonstrated the potential merits of combining low-dose methotrexate with conventional therapy, through extensively modulating the activated immune and inflammatory mediator's network, there is a need for further rigorous research of this complex network, especially involving current promising therapies which modulate this system. Such evidence has the potential to revolutionise changes for the management of this disorder. Based on the 'heterogeneity' of immune activation and inflammation among different CHF populations, an 'optimised combination treatment' may offer exciting benefits for individual therapy in the future.

Myocardial expression of the endothelin system in endotoxin-treated rats.

Although circulating plasma levels of endothelin (ET)-1 are elevated in endotoxemia, little is known about the myocardial expression of the ET system in endotoxic shock. We assessed the temporal mRNA expression pattern of key components of the ET system (pre-pro ET (ppET) -1, -2, ET-converting enzyme-1, ET(A) and ET(B) receptors) by reverse transcription polymerase chain reaction in a rat model of early endotoxic shock. Lipopolysaccharide (5 mg/kg, i.p.) caused a transient increase (p < 0.05) in inducible nitric oxide synthase mRNA expression. ppET-1 mRNA expression was increased at 2 h (approximately 12-fold increase; p < 0.05) in the lipopolysaccharide compared with the saline group and ppET-2 mRNA expression was unaltered. ET-converting enzyme-1, ET(A), and ET(B) receptor mRNA expression was unaltered in the lipopolysaccharide compared with the saline group. While ppET-1 mRNA expression is selectively upregulated in ventricular myocardium of lipopolysaccharide-treated rats, an absence of alteration in ET-converting enzyme-1 mRNA expression suggests an excess capacity of ET-converting enzyme-1 to cope with the increased expression of ET-1. At the level of the receptor, endotoxic shock did not affect the expression of either ET(A) or ET(B) receptor mRNA. These data are consistent with the increased expression of myocardial ET-1 as an acute-phase response due to hemodynamic instability associated with the early stages of endotoxic shock.

Use of A-192621 to provide evidence for involvement of endothelin ET(B)-receptors in endothelin-1-mediated cardiomyocyte hypertrophy.

Increased plasma levels of endothelin-1 correlate with the severity of left ventricular hypertrophy in vivo. The aim of the study was to determine the relative contribution of stimulation of endothelin ET(A) and endothelin ET(B) receptors, and the associated activation of protein kinase C, to the hypertrophic response initiated by endothelin-1 in adult rat ventricular cardiomyocytes maintained in culture (24 h). Endothelin-1 (10(-7) M) increased the total mass of protein and the incorporation of [14C] phenylalanine into protein to 26% and 25% greater (P<0.05) than respective basal values. The total content of RNA and the incorporation of 2-[14C] uridine into RNA were increased by 23% and 21%, respectively, by endothelin-1 (10(-8) M). Actinomycin D (5x10(-6) M), an inhibitor of transcription, abolished the incorporation of [14C] phenylalanine and the increased protein mass elicited by endothelin-1 (10(-8) M). The selective agonists at the endothelin ET(B) receptor, sarafotoxin 6c (10(-7) M) and endothelin-3 (10(-7) M), increased the incorporation of [14C] phenylalanine to 13% and 13% greater than respective basal values. The incorporation of [14C]phenylalanine in response to endothelin-1 (10(-7) M) was reduced by 50% (P<0.05) by the selective antagonist at endothelin ET(A) receptors, ABT-627 (10(-9) M), while the response to sarafotoxin 6c was not attenuated. The selective antagonist at endothelin ET(B) receptors, A192621 (10(-10) M), abolished the response to sarafotoxin 6c (10(-7) M) and attenuated the response to endothelin-1 (10(-7) M) by 43% (P<0.05). The selective inhibitor of protein kinase C, bisindolylmaleimide (5x10(-6) M) attenuated the response to sarafotoxin 6c (10(-7) M) by 78% and that to endothelin-1 (10(-7) M), elicited in the presence of A192621 (10(-10) M), by 52%. In conclusion, these data implicate endothelin ET(B) receptors, in addition to endothelin ET(A) receptors, in endothelin-1-mediated cardiomyocyte hypertrophy and provide evidence for the involvement of protein kinase C, at least in part, in the hypertrophic signalling pathways associated with activation of each receptor subpopulation.

Positive and negative contractile effects of somatostatin-14 on rat ventricular cardiomyocytes.

Somatostatin-14 elicits negative inotropic and chronotropic actions in atrial myocardium. Less is known about the effects of somatostatin-14 in ventricular myocardium. The direct contractile effects of somatostatin-14 were assessed using ventricular cardiomyocytes isolated from the hearts of adult rats. Cells were stimulated at 0.5 Hz with CaCl2 (2 mM) under basal conditions and in the presence of the beta-adrenoceptor agonist, isoprenaline (1 nM), or the selective inhibitor of the transient outward current (Ito), 4-aminopyridine (500 microM). Somatostatin-14 did not alter basal contractile response but it did inhibit (IC50 = 13 nM) the response to isoprenaline (1 nM). In the presence of 4-aminopyridine (500 microM), somatostatin-14 stimulated a positive contractile response (EC50 = 118 fM) that was attenuated markedly by diltiazem (100 nM). These data indicate that somatostatin-14 exerts dual effects directly in rat ventricular cardiomyocytes: (1) a negative contractile effect, observed in the presence of isoprenaline (1 nM), coupled to activation of Ito; and (2) a previously unreported and very potent positive contractile effect, unmasked by 4-aminopyridine (500 microM), coupled to the influx of calcium ions via L-type calcium channels. The greater potency of somatostatin-14 for producing the positive contractile effect indicates that the peptide may exert a predominantly stimulatory influence on the resting contractility of ventricular myocardium in vivo, whereas the negative contractile effect, observed at much higher concentrations, could indicate that localized elevations in the concentration of the peptide may serve as a negative regulatory influence to limit the detrimental effects of excessive stimulation of cardiomyocyte contractility.

Endothelin(A) receptor subtype mediates endothelin-induced contractility in left ventricular cardiomyocytes isolated from rabbit myocardium.

Endothelin (ET)-1 is a potent positive inotropic agent, the effects of which are mediated by increases in cytosolic Ca(2+) in the myocardium. The object of this study was to examine 1) the influence of ET(A) and ET(B) receptor subtypes, and 2) the role of the phospholipase C (PLC) pathway in mediating ET-1-induced contraction. Left ventricular cardiomyocytes were isolated from the hearts of New Zealand White rabbits (2-2.5 kg) by the use of Langendorff perfusion with collagenase. Cardiomyocyte function was examined during unloaded, electrically stimulated (0.5 Hz) contractions with a video-edge detection system. ET-1 increased cell shortening with greater potency than ET-3: mean EC(50) values were 1.1 x 10(-11) and 2.6 x 10(-10) M, respectively. With the same order of potency, ET-1 and ET-3 increased (P <.05) velocity of cell shortening. The ET(A) receptor-selective antagonist ABT-627 shifted the ET-1-induced cell shortening response curve to the right with a pA(2) value of 10.3. The ET(B) receptor-selective antagonist A-192621 (10(-8)-10(-7) M) did not alter the concentration-response of ET-1. Moreover, the ET(B) receptor-selective agonist sarafotoxin 6c did not have any effect on cell shortening over the concentration range of 10(-11) to 10(-7) M. ET-1 in the presence of the PLC inhibitor U-73122 did not alter the contractile amplitude. However, ET-1 in the presence of the protein kinase C inhibitor bisindolylmalemide increased cell shortening. These findings indicate that 1) the ET(A) receptor subtype, and not the ET(B) receptor subtype, mediates the positive inotropic effect of ET-1, and 2) the response of ET-1 is mediated by a PLC pathway, but not through protein kinase C, in ventricular cardiomyocytes isolated from rabbit myocardium.

Inotropic response to endothelin-1, isoprenaline and calcium in cardiomyocytes isolated from endotoxin treated rats: effects of ethyl-isothiourea and dexamethasone.

1. The contractile effects of endothelin-1, isoprenaline and extracellular calcium were assessed on ventricular cardiomyocytes isolated from lipopolysaccharide-treated rats. The involvement of nitric oxide was investigated using dexamethasone (in vivo) and ethyl isothiourea (in vitro). 2. Male Wistar rats (n=70) were injected with either saline (1 ml kg(-1)) or lipopolysaccharide (LPS; 5 mg kg(-1)) alone, or following pre-treatment with dexamethasone (DEX+LPS; 5 mg kg(-1)). Ventricular cell shortening was recorded using a video edge detection system, and concentration-response relationships were established for endothelin-1, isoprenaline and calcium, in the absence or presence of ethyl isothiourea (ETU; 10 microM). iNOS expression was assessed using reverse transcription-polymerase chain reaction. 3. iNOS mRNA expression was greater (P<0.001) in the LPS (iNOS/GAPDH ratio: 0.90+/-0.09) treated group compared to saline (iNOS/GAPDH ratio: 0.36+/-0.02). Baseline contractile amplitude was reduced (P<0.05) in the LPS (7.3+/-0.2 microm) and DEX+LPS groups (6.7+/-0.3 microm) compared to saline (8. 0+/-0.2 microm). 4. The concentration-dependent contractile response to endothelin-1 was attenuated (P<0.05) in the LPS group compared to saline (maximum change: 0.45+/-0.2 vs 1.8+/-0.2 microm). Neither ETU nor dexamethasone improved contractile function in the LPS-treated animals. 5. The concentration-dependent increase in the contractile response to isoprenaline was attenuated in the LPS-treated group compared to saline (P<0.05; maximum change: 1.7+/-0.4 vs 3.1+/-0.4 microm). This effect was reversed by ETU (maximum change: 3.7+/-0.6 microm). Pre-treatment with dexamethasone prevented a significant fall in contraction amplitude (maximum change: 2.4+/-0.4 microm). 6. The contractile response to calcium was reduced (P<0.05) in the LPS group compared to saline (maximum change: 8.7+/-0.6 vs 10.7+/-0.8 microm). Neither ETU nor dexamethasone restored contractile function in the LPS-treated group. 7. In conclusion, a nitric oxide-mediated inhibitory pathway is not responsible for the diminished contractile response to either endothelin-1 or extracellular calcium, but contributes to the hyporesponsiveness to isoprenaline in lipopolysaccharide treated rats.

Receptor-mediated effects of endothelin on the L-type Ca++ current in ventricular cardiomyocytes.

The purpose of this study was to establish whether specific receptor subtypes are responsible for mediating the effects of endothelin-1 (ET-1) and endothelin-3 (ET-3) on the L-type calcium current (ICa) using a number of receptor-selective antagonists, including PD155080 (ETA), BQ-788, RES-701 and IRL-1038 (ETB) and the ETA/ETB receptor-non-selective antagonist PD145065. Ventricular cardiomyocytes were isolated from adult New Zealand White rabbits using Langendorff perfusion with collagenase. ICa was recorded using a whole-cell patch-clamp technique. ET-1 decreased, whereas ET-3 increased, ICa at equimolar concentrations of 10 nM. The decrease in ICa produced by ET-1 was completely blocked by PD155080 and PD145065 (1 and 10 microM); however, ICa was increased upon washout of PD155080. Although the decrease in ICa produced by ET-1 was partially blocked by BQ-788 (1 and 10 microM), ET-1 in combination with either RES-701 (1 and 10 microM) or IRL-1038 (1 microM) produced a decrease in ICa similar to that produced by ET-1 alone. The increase in ICa by ET-3 was completely abolished by either BQ-788 or IRL-1038 (1 microM). These data indicate that the decrease in ICa produced by ET-1 in rabbit ventricular cardiomyocytes is mediated by the ETA receptor subtype, because PD155080 completely inhibited this response. The ETB receptor-selective antagonists RES-701 and IRL-1038 did not alter the decrease in current produced by ET-1, although the response was partially sensitive to BQ-788, which may lack receptor-subtype selectivity in these cells. In contrast, the increase in ICa produced by ET-3 was mediated by the ETB receptor subtype, because BQ-788 and IRL-1038 abolished this response.

Stimulation of L-type Ca2+ current by the endothelin receptor A-selective antagonist, BQ-123 in ventricular cardiomyocytes isolated from the rabbit myocardium.

BQ-123 is extensively used as an antagonist at endothelin (ET) receptors, having selectivity at the ET(A) receptor subtype. In this study, the effects of BQ-123 per se on action potentials, L-type calcium currents, and potassium currents, were examined in ventricular cardiomyocytes isolated from adult, male, New Zealand White rabbits, using the patch-clamp technique. BQ-123 (1 microM) increased (P < 0.02) the duration of the action potential to 267 +/- 36 ms from a control duration of 228 +/- 30 ms. BQ-123 did not have any effect on the inward rectifier or transient outward potassium currents, but increased (P < 0.02) the L-type Ca2+ current to -2.76 +/- 0.3 nA from a control value of -2.45 +/- 0.28 nA. The increases in both duration of the action potential and L-type Ca2+ current were reversed upon washout (233 +/- 28 ms and -2.32 +/- 0.31 nA, respectively) and were not different from the control values in the absence of BQ-123. In contrast, the endothelin receptor antagonists, BQ-788, PD155080 and PD145065 (1-10 microM) did not affect the L-type Ca2+ current. These results indicate that, unlike PD155080, BQ-788 and PD145065, the conventional ET(A) receptor-selective antagonist, BQ-123, exerts a unique positive effect on the L-type Ca2+ current in ventricular cardiomyocytes isolated from rabbit myocardium. The mechanism of action of BQ-123, therefore, is not confined to ET receptor antagonism.

Economic evaluation of the use of nadroparin calcium in the prophylaxis of deep vein thrombosis and pulmonary embolism in surgical patients in Italy.

The objective of this study was to compare the costs, from the perspective of the payer, of using nadroparin calcium, a low-molecular-weight heparin, instead of unfractionated heparin in the prophylaxis of venous thromboembolism in patients undergoing orthopaedic surgery or major general surgery in Italy. The methods used were based on a published meta-analysis and a survey of clinical practice. We constructed a model of the prophylaxis and management of venous thromboembolism in Italy. Resource use associated with individual events was estimated on the basis of the clinical survey. Unit costs, not available from published sources, were taken from charges made by hospitals and from direct observation. A sensitivity analysis was conducted to examine whether the results were robust to changes in key variables. In the base case, compared with unfractionated heparin, prophylaxis with nadroparin calcium reduced the expected costs of managing thromboembolism by 267,226 Italian lire (L, 1994 values; $US1 = L1600 approx.) per patient undergoing orthopaedic surgery, and by L45,588 per patient undergoing major general surgery. Therefore, switching from unfractionated heparin to nadroparin calcium in these patients offers the possibility of significant cost savings to the Italian healthcare system.

Economic evaluation of the use of nadroparin in the treatment of deep-vein thrombosis in Switzerland.

OBJECTIVE: To compare the cost implications, from the payer's perspective, of the use of nadroparin instead of unfractionated heparin in the initial treatment of deep-vein thrombosis. DESIGN: Cost-minimization study. SETTING: Switzerland. MATERIAL: Survey of clinical practice in six Swiss hospitals used to model three treatment regimens. MAIN OUTCOME MEASURES: Cost of treatment ($ US) per patient. RESULTS: Treatment with nadroparin instead of unfractionated heparin would reduce costs by $153 per patient. Treatment with nadroparin instead of subcutaneous unfractionated heparin would reduce costs by $109 per patient. CONCLUSIONS: The cost of initial treatment of deep-vein thrombosis is considerably lower with nadroparin than with either of the alternative regimens. Nadroparin reduces costs through greater ease of administration and by reducing the amount of laboratory monitoring. Treatment with nadroparin might also allow patients to be discharged from the hospital more quickly than is possible with intravenous infusion of unfractionated heparin.

Involvement of endothelin (ET)A and ETB receptors in the hypertrophic effects of ET-1 in rabbit ventricular cardiomyocytes.

The question was addressed whether endothelin-1 (ET-1) exerts hypertrophic effects in cardiomyocytes isolated from ventricles of adult rabbits and maintained in short-term (24 h) serum-free primary culture providing mechanical quiescence. ET-1 (> or =100 pM) increased significantly total mass of cellular protein and incorporation of L-U-[(14)C]phenylalanine and 2-[(14)C]uridine into cellular protein and RNA, respectively. Cycloheximide (35 microM), an inhibitor of protein synthesis, significantly reduced the incorporation of L-U-[(14)C]phenylalanine and 2-[(14)C]uridine into cellular protein and RNA, respectively, under control conditions and in response to ET-1. Actinomycin D (5 microM), a selective inhibitor of transcription, abolished the incorporation of 2-[(14)C]uridine into cellular RNA and significantly reduced the incorporation of L-U-[(14)C]phenylalanine into cellular protein under control conditions and in response to ET-1. The selective antagonists at the ET(A) receptor [BQ123 (100 nM) and PD155080 (100 nM)] and the selective antagonist at the ET(B) receptor [BQ788 (100 nM)] significantly reduced the incorporation of L-U-[(14)C]phenylalanine into cellular protein in response to ET-1 (10 nM). The selective inhibitor of protein kinase C (PKC), bisindolylmaleimide (BIM) (5 microM), reduced markedly the incorporation of 2-[(14)C]uridine into cellular RNA and, to a lesser degree, the incorporation of L-U-[(14)C]phenylalanine into cellular protein in response to ET-1 (100 pM to 10 nM). ET-1 exerts hypertrophic effects directly in vitro in ventricular cardiomyocytes isolated from the hearts of adult rabbits. These effects are (a) due to de novo synthesis since total mass of cellular protein and incorporation of L-U-[(14)C]phenylalanine and 2-[(14)C]uridine into cellular protein and RNA, respectively, were increased; (b) mediated by both the ET(A) and ET(B) receptor subtypes; and (c) may be associated, at least partly, with the activation of PKC.

Characterisation of a cellular model of cardiomyopathy, in the rabbit, produced by chronic administration of the anthracycline, epirubicin.

The objective of this study was to assess the structural, mechanical and electrophysiological changes associated with chronic administration of epirubicin (Pharmorubicin), which is a cardiotoxic anthracycline antibiotic used in conventional cancer chemotherapy. New Zealand white rabbits (8 weeks) were treated with twice-weekly infusions of epirubicin (2 mg/kg) or saline for a period of 6 weeks, followed by a wash-out period of 2 weeks. Myocardial damage consistent with cardiomyopathy was observed in the epirubicin-treated animals; electron micrographs indicated myofibril loss together with separation of the intercalated disc and dilation of the sarcotubular system. Contractile function, as measured by mechanical shortening, action potentials and L-type Ca2+ currents were examined in ventricular cardiomyocytes, which were isolated by means of enzymatic dissociation using collagenase. There was an attenuation in the contractile response to isoprenaline in cardiomyocytes isolated from the hearts of epirubicin-treated rabbits compared to control rabbits. Cardiomyocytes isolated from epirubicin-treated rabbits had greater basal contractile amplitude (11.0+/-0.3 %dL, n=8) than control myocytes (8.2+/-0.3 %dL, n=9), but had similar maximum responses of 19.1+/-0.6 %dL, and 17.3+/-0.5 %dL, respectively, when stimulated with 1 microM isoprenaline. No differences were noted in the peak L-type Ca2+ current of myocytes isolated from the hearts of control and epirubicin-treated rabbits; however, in the latter, there was a prolongation of the action potential duration (396+/-25 ms) compared to that in controls (321+/-26 ms). These results demonstrate structural and mechanical alterations in ventricular cardiomyocytes which are compatable with a mild cardiomyopathy following chronic treatment with the anthracycline, epirubicin. The increase in basal contraction is likely to be due to more efficient coupling of electrical stimulation, and the depressed inotropic responsiveness following stimulation with isoprenaline indicates that there may be changes in cell membrane properties. Compared to control cardiomyocytes, cells isolated from the hearts of epirubicin-treated rabbits were more heterogeneous, with respect to cell dimensions, and had significantly different electromechanical properties.

Endothelin-1 mediated inhibition of the acetylcholine-activated potassium current from rabbit isolated atrial cardiomyocytes.

1. Endothelin-1 is a 21 amino acid peptide with potent inotropic and chronotropic actions in the heart. Relatively little is known about the underlying electrophysiological effects of the peptide. In this study, the effects of endothelin-1 (ET-1) on the acetylcholine-activated potassium current (IK(ACh) were investigated in the absence and presence of the receptor-selective antagonists, PD155080 (ETA receptor-selective) and RES-701 (ETB receptor-selective) in rabbit atrial cardiomyocytes. 2. Cells were obtained from New Zealand White rabbits (2.5-3 kg) by enzymatic dissociation with collagenase. Potassium currents were recorded, in the presence of nifedipine (5 microM), by use of the whole cell ruptured patch-clamp technique. Following stabilization, control recordings were made with standard pulse protocols, and drugs were applied by a gravity fed microperfusion system. 3. Endothelin-1 (10 nM) alone did not affect the "steady state' potassium current. Acetylcholine (1 microM) increased (P < 0.05) the potassium current to-1321 +/- 290 pA, from a control value of -955 +/- 191 pA, at a step potential of -100 mV. Acetylcholine also increased the holding current at -40 mV from +80 +/- 9 pA to +242 +/- 38 pA, and this effect was abolished (P < 0.05) in the presence of endothelin-1 (+44 +/- 13 pA). The responses to acetylcholine were attributed to activation of the atrial muscarinic-activated potassium current (IK(ACh)) as they were blocked by atropine (10 microM). Endothelin-1 (10 nM) in the presence of acetylcholine did not affect the "steady state' potassium current (-882 +/- 88 pA compared to a control value of -870 +/- 98 pA, at -100 mV). 4. The ETA receptor-selective antagonist, PD155080 (1 microM), prevented (P < 0.05) the ET-1 induced inhibition of IK(ACh) at all potentials. PD155080, in the presence of endothelin-1 and acetylcholine, increased the inward component of the "steady state' potassium current to -1030 +/- 210 pA from a control value of -804 +/- 224 pA at a step potential of -100 mV. Also the outward component was increased at a potential of -20 mV from +90 +/- 17 pA to +241 +/- 47 pA. 5. Unlike PD155080, the ETB receptor-selective antagonist, RES-701 (1 microM), only prevented (P < 0.05) the inhibitory effect of endothelin-1 on the inward component of the IK(ACh); at -100 mV, RES-701, in the presence of endothelin-1 and acetylcholine, increased the "steady state' potassium current to -913 +/- 137 pA from -733 +/- 116 pA. Furthermore, RES-701, in contrast to PD155080, failed to sustain this inhibitory effect as, in the presence of endothelin-1 and acetylcholine, the "steady state' potassium current returned to a value of -768 +/- 96 pA, at a step potential of -100 mV. 6. In conclusion, endothelin-1 clearly inhibits the effects of acetylcholine on IK(ACh) in rabbit atrial cardiomyocytes. This effect is primarily mediated by an ETA receptor-subtype, but is transiently and partially mediated by a RES-701-sensitive ETB receptor subtype. Inhibition of the IK(ACh) may account for the positive chronotropic properties of endothelin-1.

Mechanical effects of ET-1 in cardiomyocytes isolated from normal and heart-failed rabbits.

Endothelin (ET-1) is found at elevated concentrations in the plasma of patients with heart failure and in animal models of cardiomyopathy. The peptide is a potent positive inotropic agent, the effects of which are mediated by increases in cytosolic Ca2+ in cardiomyocytes. The object of this study was to investigate at the cellular level, the actions of ET-1 on contractile function and on Ca2+ currents in heart-failed ventricular myocardium. Male New Zealand White rabbits (8 wks) were treated with twice weekly injections of epirubicin (4 mg/kg/wk, n = 7) or with saline (n = 7) for 6 wks, followed by a washout period of 2 wks. Ventricular cardiomyocytes were isolated from rabbit hearts using Langendorff perfusion with collagenase; contractile function was examined using a video microscopy method, and L-type Ca2+ currents were recorded using a whole-cell patch-clamp technique. ET-1 produced a concentration-dependent increase in contractile response (% increase from basal value) to a maximum at 1 nM ET-1 of 69 +/- 11% (mean +/- S.D.) in control cardiomyocytes and 33 +/- 6% in heart-failed cells. However, there was no significant change in the EC50 obtained with ET-1 for healthy (0.31 +/- 0.1 nM) and for failed cardiomyocytes (0.24 +/- 0.1 nM). The effects of ET-1 on L-type Ca2+ channels were similar with a peak amplitude at 1 nM ET-1 of -3.26 +/- 0.8 nA in control cardiomyocytes and -3.32 +/- 0.9 nA in heart-failed cells. The attenuation of the contractile response to ET-1 in heart-failed cells may reflect a desensitization of ET receptors as a consequence of elevated circulating levels of ET and was not reflected by alteration of transmembrane Ca2+ conductance. It is probable, therefore, that multiple signalling pathways are involved in the actions of ET on ventricular myocardium.

Actions of the novel vasodilator, flosequinan, in isolated ventricular cardiomyocytes.

Although the potent vasodilating effect of flosequinan is well characterised, the positive inotropic action reported is more varied and less well understood. We examined the contractile and electrophysiologic effects of flosequinan and its metabolite, BTS 53554, in cardiomyocytes from either adult male Sprague-Dawley rats (200-250 g) or New-Zealand White rabbits (2-2.5 kg) and compared the effects with those of sulmazole and enoximone [selective phosphodiesterase (PDE) III inhibitors], Ro 20-1724 and rolipram (selective PDE IV inhibitors) and 3-isobutyl-1-methylxanthine (IBMX, nonselective PDE inhibitor). Flosequinan and BTS 53554 had positive contractile effects (p < 0.05) in both rat and rabbit ventricular cardiomyocytes only at the maximum concentration (10(-3) M). Differences were noted between species, however. Flosequinan 10(-3) M had a greater contractile effect than BTS 53554 (10(-3) M) in rabbit cardiomyocytes, but not in rat cardiomyocytes. We studied the interaction of flosequinan or the metabolite with other PDE inhibitors in rat cardiomyocytes. Contractile amplitudes were not significantly different with equimolar concentrations (3 x 10(-4) M) of Ro 20-1724, flosequinan, or BTS 53554 alone (15 +/- 6, 18 +/- 4, and 32 +/- 10%, respectively, greater than the mean basal dL value of 7.38 +/- 0.12%, mean +/- SE error). However, the combinations of Ro 20-1724 with flosequinan and Ro 20-1724 with BTS 53554 produced synergistic responses: 71 +/- 10 and 72 +/- 14%, respectively, greater than the mean basal dL value (p < 0.05). In contrast, the combinations of either flosequinan or BTS 53554 with IBMX or sulmazole produced no further increase in contractile amplitude. Neither flosequinan nor BTS 53554 produced any detectable increase in cyclic AMP, whereas significant increases were noted with Ro 20-1724, IBMX, and sulmazole (p < 0.05) in rat cardiomyocytes. Flosequinan increased beating frequency in rat isolated right auricles concentration dependently and was significant over the concentration range of 10(-5)-3 x 10(-4) M; flosequinan 3 x 10(-4) M maximally increased the mean frequency of beating by 35% of the predrug value (255 +/- 15 beats/min). Flosequinan had no effect on resting membrane potential, amplitude, or maximum upstroke velocity in rat isolated left ventricular (LV) papillary muscle, but at the maximum concentration (10(-3) M), flosequinan decreased action potential duration (APD) at 10, 50, and 75% of repolarization (p < 0.05). BTS 53554 produced no changes in AP characteristics over the concentration range of 10(-5)-10(-3) M.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential effects of phosphodiesterase inhibitors on accumulation of cyclic AMP in isolated ventricular cardiomyocytes.

The intracellular actions of phosphodiesterase (PDE) inhibitors on the accumulation of cyclic nucleotides were studied in isolated ventricular cardiomyocytes from adult Sprague-Dawley rats. Elevated levels of cyclic AMP, due to the effects of selective PDE inhibitors, were detected only when the levels of cyclic nucleotide were enhanced with forskolin (10 microM). The time course for the elevation of cyclic AMP levels was similar for all the PDE inhibitors tested, following the pattern of an initial rise in the first 2-4 min, proceeded by a steady state at 67 +/- 6% of the maximum stimulation. HN-10200 (2-[3-methoxy-5-methylsulfinyl-2-thienyl]-1H-imidazo-[4,5-c]- pyridine hydrochloride), a new imidazopyridine derivative, had a similar concentration-dependent profile to the structurally related compound, sulmazole (AR-L 115 BS, 2-[2-methoxy-4-methylsulfinyl)phenyl]-1H- imidazo-[4,5-b]-pyridine). Both the non-selective inhibitor, 3-isobutyl-1-methylxanthine (IBMX), and the selective PDE IV inhibitor, Ro 20-1724 (4-[(3-butoxy-4-methoxyphenyl)methyl]-2- imidazolidinone), potentiated the forskolin-stimulated levels of cyclic AMP with a much greater efficacy than sulmazole or HN-10200. The concentrations of forskolin required by IBMX, sulmazole and HN-10200 (10(-3) M) to increase levels of cyclic AMP by 4 pmol/mg protein were 3.2 x 10(-6) M, 1.32 x 10(-5) M and 1.46 x 10(-5) M, respectively. Enoximone failed to cause an increase in the levels of cyclic AMP, even when stimulated with maximal concentrations of forskolin. Furthermore, in the presence of forskolin, enoximone attenuated the response of Ro 20-1724 and IBMX in a concentration-dependent manner. Enoximone, similarly to HN-10200, sulmazole, Ro 20-1724 and IBMX did not produce any significant effect on levels of cyclic GMP under elevated conditions in the presence of sodium nitroprusside. The combined action of Ro 20-1724, with either HN-10200, sulmazole, or IBMX (10(-4) M), on intracellular levels of cyclic AMP, was not greater than the response to Ro 20-1724 alone. These data demonstrate the differential actions of PDE III and PDE IV inhibitors in rat ventricular cardiomyocytes. It is suggested that enoximone has a high selectivity for the PDE III isoenzyme so that hydrolysis of cyclic AMP by the PDE IV isoenzyme is not inhibited, in accordance with the lack of increase in cyclic AMP by enoximone in rat cardiomyocytes. HN-10200 and sulmazole, producing small increases in intracellular levels of cyclic AMP, are less selective PDE III inhibitors than enoximone.(ABSTRACT TRUNCATED AT 400 WORDS)

Cardiotonic actions of selective phosphodiesterase inhibitors in rat isolated ventricular cardiomyocytes.

1. The contractile effects of the novel cardiotonic agent HN-10200 (2-[3-methoxy-5-methylsulphinyl-2-thienyl]-1H-imidazo-[4,5-c]-p yri dine hydrochloride), were examined and comparisons made with the responses obtained to a structurally similar compound, sulmazole, and to a number of other compounds which are known to inhibit phosphodiesterase (PDE) isoenzymes with differing selectivities; namely, enoximone (PDE III inhibitor), Ro 20-1724 (PDE IV inhibitor) and 3-isobutyl-1-methylxanthine (non-selective PDE inhibitor). 2. Contractile function, as measured by mechanical shortening, and biochemical systems involving cyclic AMP were investigated in ventricular cardiomyocytes isolated from adult Sprague-Dawley rats (200-250 g). 3. HN-10200 exerted a concentration-dependent (10(-8) M-10(-4) M) positive contractile effect, which was independent of alpha- or beta-adrenoceptor, or histamine receptor stimulation. 4. The efficacies of the contractile responses to the PDE inhibitors were of the order: HN-10200 > IBMX > sulmazole > enoximone and maximum stimulations, which were obtained at concentrations of 10(-4) M, were 54 +/- 4%, 41 +/- 7%, 38 +/- 7% and 26 +/- 5% (mean +/- s.e.) greater than basal levels, respectively (n = 6); the basal value of contractile amplitude (dL), in the absence of PDE inhibitors was 7.39 +/- 0.18% (mean +/- s.e.). Ro 20-1724 did not have any effect on contractile activity. 5. Due to low basal levels of cyclic nucleotides in isolated cells, accumulation of cyclic AMP due to the presence of the PDE inhibitors was detected only when the levels of cyclic nucleotide were enhanced with forskolin (10 microM). 6. The PDE inhibitors increased levels of cyclic AMP only at concentrations> 10-4 M. HN-10200 and sulmazole had similar concentration-dependent profiles for the accumulation of cyclic AMP; their potencies were lower than that of IBMX (concentrations of forskolin required to increase cyclic AMP by 4 pmol mg-1 protein, in the presence of maximum concentrations of the PDE inhibitors, were 13 +/- 311M, 14 +/- 3 JAM and 3 +/- 0.6 JAM [mean +/- s.e.], respectively).7. These results indicate that a similar mechanism, probably through a weak inhibition of the cyclic AMP-specific PDE isoenzymes, is responsible for the increase in levels of cyclic AMP by HN-10200 and sulmazole. However, cyclic AMP is only partially responsible for the positive contractile effect of HN-10200 and, similarly, sulmazole and IBMX. The lack of apparent increase in levels of cyclic AMP by enoximone, highlights its degree of selectivity for the PDE III isoenzyme, such that the PDE IV isoform is still present in sufficient quantity to degrade cyclic AMP within the cell. On the other hand,the potent action of Ro 20-1724 on accumulation of cyclic AMP, in addition to the lack of effect on contractile function, is in agreement with the selectivity of this compound for the PDE IV isoenzyme and compartmentalization of cyclic AMP in rat isolated ventricular cardiomyocytes.