Expression and Hsp27, troponin T and troponin I co-localization in cardiac tissue after naloxone-precipitated morphine withdrawal

E Martinez-Laorden, ML Laorden, MV Milanes, P Almela. University of Murcia, Pharmacology 30100, Spain

Introduction

Like stressors, morphine withdrawal leads to an increase in circulating catecholamines which act through beta-adrenergic receptors augmenting consumption of oxygen and inducing free oxygen radicals that impart a negative influence on the biochemical status of the cell. Cellular stress can cause a sudden change in the cellular environment, to which the cell is not prepared to respond and there is an urgent need of cellular safeguards in the form of heat shock proteins (Hsps). Hsp27 has been shown to protect the heart against contractile dysfunction via stabilizing myofilaments, in particular troponin TnI and TnT, two elements of the Tn complex that, in concert with tropomyosin, regulates myocyte contraction in response to a rise of intracellular Ca2+ concentration.

In this study, we have investigated Hsp27 phosphorylation at Ser82 (p-Hsp27) during morphine dependence and withdrawal and have evaluated the interaction between Hsp27 and TnT and TnI in the heart.

Material and Methods

Dependence on morphine was induced by a 6-days s.c. implantation of morphine pellets in male Sprague-Dawley rats (220-240 g). Morphine withdrawal was precipitated on day 7 by injection of naloxone (2 mg/kg, s.c.). Rats were killed 60 minutes after naloxone injection. Hsp27 phosphorylation at Ser82 as well as TnT and TnI expression was determined by western blot. Hsp27-TnT and Hsp27-TnI co-localization was studied by immunofluorescence. Results are expressed as the mean ± SEM. Data were analyzed by analysis of variance (ANOVA) followed by the Newman-Keuls post-hoc test.

Results and Conclusions

Our results show that naloxone-precipitated morphine withdrawal increased pHsp27 (187 ± 19.6, n = 5) and TnT (164.1 ± 16.07, n = 4) expression 60 min after the injection of the opioid antagonist. However, there were not changes in these proteins in the morphine dependent group injected with saline (88.39 ± 8.27, n = 5; 157 ± 19.35, n = 4, respectively). By contrast, we observed a decrease in TnI (71.21 ± 11.19, n = 4) expression during morphine withdrawal compared to its control group (90.42 ± 10.80, n = 4).

Secondly, we visualized Hsp27, TnI or TnT immunofluorescence in cardiac tissue and observed a specific Hsp27-TnT and Hsp27-TnI co-localization. Present findings demonstrated that morphine withdrawal is capable of inducing Hsp27 activation in cardiac tissue and that this protective effect is mediated by TnT and TnI myofilaments.