Key words: endotoxin, interferon- , isoprenaline, lipocortin-. 1, papillary muscle. INTRODUCTION. In humans, depression of myocardial contractile function is ...
Clinical and Experimental Pharmacology and Physiology (1999) 26, 522–524
SHORT COMMUNICATION
LIPOCORTIN-1 PRESERVES MYOCARDIAL RESPONSIVENESS TO b-ADRENERGIC STIMULATION IN RAT PAPILLARY MUSCLE Rebecca H Ritchie, Xiaolu Sun and Gregory J Dusting Howard Florey Institute of Experimental Physiology and Medicine, The University of Melbourne, Parkville, Victoria, Australia
SUMMARY 1. During septic shock, myocardial contractile dysfunction is accompanied by the release of cytokines and enhanced production of nitric oxide, and the contractile dysfunction is prevented by glucocorticoids. 2. Myocardial dysfunction was induced in vitro by incubation of rat papillary muscle for 15 h with endotoxin (lipopolysaccharide, LPS) and interferon-g (IFN-g). 3. Both baseline contractile function and inotropic responsiveness to isoprenaline were markedly reduced by the combination of LPS plus IFN-g. 4. Lipocortin-1 (LC-1) is induced by glucocorticoids, and LC-12–26, its N-terminal fragment, protected the papillary muscle inotropic responsiveness to isoprenaline, but did not affect the decline in baseline contractile function induced by LPS plus IFN-g. 5. The mechanisms of this protective action need to be explored further, but LC-1 may prove to be a novel cardioprotective agent for the management of septic shock. Key words: endotoxin, interferon-g, isoprenaline, lipocortin1, papillary muscle.
INTRODUCTION In humans, depression of myocardial contractile function is observed in both septic shock and administration of endotoxin (the lipopolysaccharide component of the bacterial cell wall).1,2 This myocardial depression is often refractory to b-adrenergic agonist administration3 and is associated with induction of nitric oxide synthase 2 (NOS2), the inducible isoform of NOS.4 In vitro incubation of isolated cardiac myocytes with lipopolysaccharide (LPS) and cytokines also elicits NOS2 induction.5,6 These conditions reduce baseline contraction in addition to depressing inotropic responsiveness to isoprenaline in rat papillary muscles and Correspondence: Associate Professor GJ Dusting, Howard Florey Institute of Experimental Physiology & Medicine, The University of Melbourne, Parkville, Victoria, Australia. Email: Presented at the 20th Annual Scientific Meeting of the High Blood Pressure Research Council of Australia, Melbourne, December 1998. This paper has been peer reviewed. Received 14 December 1998; revision 17 February 1999; accepted 24 February 1999.
whole hearts: both effects are prevented by the glucocorticoid dexamethasone.6,7 Glucocorticoids induce synthesis and release from many cell types of lipocortin-1 (LC-1),8,9 a member of the annexin family. However, the contribution of LC-1 to the effects of dexamethasone in the heart, in terms of NOS2 induction or the prevention of cardiac depression, has not been elucidated. Therefore, we examined the effects of the N-terminus peptide LC-12–26 fragment on myocardial dysfunction induced by a combination of LPS plus interferon (IFN)-g in papillary muscles.
METHODS Isolated papillary muscle preparations Papillary muscles were isolated7 from the left ventricle of male SpragueDawley rat hearts (250–400 g) under ether/oxygen anaesthesia and were attached by silk thread to a Grass FT03C force displacement transducer as previously described.7 The tissue baths contained Krebs’ buffer (composition: NaCl 118; KCl 4.7; CaCl2 2.5; MgSO4 1.2; NaHCO3 25; KH2PO4 1.2; glucose 11 mmol/L) gassed with 95 : 5% O2 : CO2 throughout the experiment to maintain pH 7.4. Tissue bath temperature was maintained at 30°C. Muscles were electrically driven by Grass S-44 stimulators at 0.2 Hz. Rectangular pulses 5 msec in duration and 20% above threshold (< 20 V) were delivered through a pair of longitudinal platinum electrodes in close proximity to the muscles. Isometric developed tension and its derivative were recorded with a Maclab computer (ADI, Sydney, NSW, Australia). Muscle length was maintained at maximum active tension for the duration of the experiment.
Experimental protocol Peak isometric developed tension at baseline was recorded after equilibration. Cumulative concentration–response curves to isoprenaline (0.001– 10 mmol/L) were generated for peak developed tension. Thirty minutes after washout of isoprenaline, papillary muscles were incubated for 15 h in the presence and absence of IFN-g (100 U/mL) plus LPS (100 mg/mL), prior to construction of a second isoprenaline concentration–response curve. The influence of LC-12–26 (1 mmol/L) added 30 min before IFN-g + LPS on both peak tension at baseline and responsiveness to isoprenaline was determined.
Materials Endotoxin (LPS), recombinant murine IFN-g and isoprenaline were obtained from Sigma (St Louis, MO, USA). Ac-Ala2-lipocortin-1 (LC-12–26, Ac-AlaMet-Val-Ser-Glu-Phe-Leu-Lys-Gln-Ala-Trp-Phe-IIe-Glu-Asn-Glu-Glu-GlnGlu-Tyr-Val-Gln-Thr-Val-Lys-OH) was obtained from Bachem AG (Bubendorf, Switzerland).
Lipocortin-1 preserves b-adrenergic contractility
Statistics Baseline contractile responses (peak tension) were presented as mean 6 SEM. Responses to isoprenaline challenge were expressed as a percentage of the maximum response to the first isoprenaline challenge. Student’s paired t-test was used for intra-group comparisons. Inter-group comparisons were performed using one-way analysis of variance (ANOVA), followed by Fisher’s exact test. A value of P < 0.05 was considered significant.
RESULTS Fifteen hours incubation of papillary muscles with IFN-g + LPS reduced peak tension at baseline from 0.1360.03 to 0.0760.02 g (n = 5, P < 0.05). Pretreatment with LC-12–26 did not prevent this decline: peak tension decreased from 0.1560.01 to 0.0860.01 g (n = 4, P < 0.05). Incubation of papillary muscles with IFN-g plus LPS for 15 h significantly decreased the sensitivity and the magnitude of the response of muscles to isoprenaline. Co-incubation with LC-12–26 prevented this reduction in sensitivity (Fig. 1). During construction of isoprenaline concentration–response curves, the maximum developed tension after IFN-g + LPS was reduced to 2866% of that observed initially; with LC-12–26 pretreatment this was restored to 9862% of the maximum control response (n = 4, P < 0.05 vs IFN-g + LPS alone).
DISCUSSION Glucocorticoids induce in many cell types the production of LC-1,10,11 and this in turn modulates arachidonic acid release11 and inhibits neutrophil migration,11,12 which is indicative of a second messenger role as an anti-inflammatory mediator. In the present study, the active LC-1 terminal peptide prevented the reduction in b-adrenergic responsiveness induced by 15 h incubation with IFN-g + LPS, although it did not affect the decline in baseline contractions. We have previously observed that the glucocorticoid dexamethasone protects papillary muscles from both the depression of baseline contractile function and inotropic responsiveness to b-adrenergic stimulation.7 Taken together, these results suggest that
Fig. 1 Peak isometric tension concentration–response curves to isoprenaline (0.001–10 mmol/L) in isolated rat papillary muscles before (s) and after 15 h incubation with interferon-g + lipopolysaccharide, in the absence < ). The vertical bars represent (d) and in the presence of lipocortin-1 (LC-1, s SEM and are smaller than the symbols in some cases.
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LC-1 might mediate the glucocorticoid effects on b-adrenergic responsiveness, but not the failure of baseline contractility induced by cytokines. The impairment of b-adrenergic responsiveness induced by cytokines and endotoxin appears to be mediated, at least in part, by induction of NOS2;6,7,13 dexamethasone may restore contractile responsiveness (both at baseline and with isoprenaline) by inhibition of NOS2 induction. The mechanisms by which glucocorticoids attenuate cytokine-induced NOS2 expression are multiple and include interruption of selected components of the immune response that triggers NOS2 induction and suppression of NOS2 expression.14,15 An additional mechanism of glucocorticoid action is modulation of expression of a large number of enzymes and other proteins.16 The understanding of the relative contribution of these different mechanisms and how this relates to the therapeutic effects of glucocorticoid therapy is of considerable clinical importance. In macrophages, LC-1 has been reported to mediate the inhibition by dexamethasone of NOS2 induction by endotoxin;17 it remains to be determined whether LC-1 mediates its protective effect on the myocardium via inhibition of the induction of NOS2 mRNA and protein. In conclusion, LC-1 prevents the inhibition of badrenergic responsiveness by endotoxin and cytokines, but had no effect on the baseline contractions. Although the specific mechanism of its protective effect on the heart remains to be determined, LC-1 may prove to be a novel cardioprotective agent for the treatment of myocardial depression.
ACKNOWLEDGEMENTS This study was supported by the National Health and Medical Research Council, the National Heart Foundation and a student scholarship to Xiaolu Sun from AusAid. We are grateful for advice from Professor Rod Flower of the William Harvey Research Institute (UK).
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