Global changes in the rat heart proteome induced by prolonged morphine treatment and withdrawal

Abstract

Morphine belongs among the most commonly used opioids in medical practice due to its strong analgesic effects. However, sustained administration of morphine leads to the development of tolerance and dependence and may cause long-lasting alterations in nervous tissue. Although proteomic approaches enabled to reveal changes in multiple gene expression in the brain as a consequence of morphine treatment, there is lack of information about the effect of this drug on heart tissue. Here we studied the effect of 10-day morphine exposure and subsequent drug withdrawal (3 or 6 days) on the rat heart proteome. Using the iTRAQ technique, we identified 541 proteins in the cytosol, 595 proteins in the plasma membrane-enriched fraction and 538 proteins in the mitochondria-enriched fraction derived from the left ventricles. Altogether, the expression levels of 237 proteins were altered by morphine treatment or withdrawal. The majority of changes (58 proteins) occurred in the cytosol after a 3-day abstinence period. Significant alterations were found in the expression of heat shock proteins (HSP27, α-B crystallin, HSP70, HSP10 and HSP60), whose levels were markedly up-regulated after morphine treatment or withdrawal. Besides that morphine exposure up-regulated MAPK p38 (isoform CRA_b) which is a well-known up-stream mediator of phosphorylation and activation of HSP27 and α-B crystallin. Whereas there were no alterations in the levels of proteins involved in oxidative stress, several changes were determined in the levels of pro- and anti-apoptotic proteins. These data provide a complex view on quantitative changes in the cardiac proteome induced by morphine treatment or withdrawal and demonstrate great sensitivity of this organ to morphine.

Figure 1. Flow diagram of the fractionation procedure for the rat myocardium.

The left ventricles were cut into small pieces with dissecting scissors, homogenized using an Ultra-Turrax homogenizer and subsequently with a motorized glass-Teflon homogenizer. The postnuclear supernatant (PNS I) after low-speed centrifugation was collected and the pellet rehomogenized in a glass-Teflon homogenizer. The resulting postnuclear supernatant (PNS II) was collected, mixed with PNS I and applied on the top of 18% Percoll density gradient. After high-speed centrifugation, the top portion of the gradient mainly containing soluble material was further centrifuged to yield a clear cytosolic fraction. The upper layer enriched in plasma membranes (PM) and the lower layer rich in mitochondria (MT) were separately diluted in TME buffer and spun down to remove the Percoll, which formed a compact glassy pellet at the bottom of the centrifuge tube. The sedimented PM or MT containing material formed a loose white layer on the Percoll pellet and could be easily collected by pipetting. All centrifugation steps were performed at 4°C and tubes were kept on ice during samples collection.

Figure 2. Classification of proteins identified in the left ventricular myocardium according to their subcellular localization and function.

The proteins of each fraction (CT, PM and MT) isolated from hearts of control (C), morphine-treated (M), and morphine-withdrawn (M_W-I and M_W-II) rats were labeled by isobaric reagents provided in iTRAQ 4-plex reagent kit. After LC-MALDI analyses performed on Ultimate 3000 HPLC system and acquisition of spectra on 4800 Plus MALDI TOF/TOF analyzer, proteins were identified and quantified using Protein Pilot 4.0. Localization and function of the proteins were assigned on the basis of current annotations in the Swiss-Prot database. Sections of the pie charts represent the proportion of proteins found within each functional category.

Figure 3. Venn diagrams showing the distribution of myocardial proteins in the cytosolic fraction altered by morphine treatment and withdrawal.

Numbers in the individual circles or sectors refer to the number of altered proteins which were determined in the cytosolic fraction isolated from the left ventricles of morphine-treated (M) and morphine-withdrawn for 3 days (M_W-I) or 6 days (M_W-II) rats. These proteins were arranged according to their function into 16 groups.

Figure 4. Venn diagrams showing the distribution of myocardial proteins in the plasma membrane-enriched fraction altered by morphine treatment and withdrawal.

Numbers in the individual circles or sectors refer to the number of altered proteins which were determined in the PM-enriched fraction isolated from the left ventricles of morphine-treated (M) and morphine-withdrawn for 3 days (M_W-I) or 6 days (M_W-II) rats. These proteins were arranged according to their function into 16 groups.

Figure 5. Venn diagrams showing the distribution of myocardial proteins in the mitochondria-enriched fraction altered by morphine treatment and withdrawal.

Numbers in the individual circles or sectors refer to the number of altered proteins which were determined in the MT-enriched fraction isolated from the left ventricles of morphine-treated (M) and morphine-withdrawn for 3 days (M_W-I) or 6 days (M_W-II) rats. These proteins were arranged according to their function into 16 groups.

Figure 6. Immunoblot analyses of the unphosphorylated and phosphorylated forms of HSP27.

Cytosolic proteins (20 µg) isolated from the left ventricular myocardium of control (C), morphine-treated (M), and morphine-withdrawn for 3 days (M_W-I) or 6 days (M_W-II) rats were separated on 15% polyacrylamide gels by SDS-PAGE and electrotransferred onto the nitrocellulose membrane. Specific primary antibodies were used for the detection of unphosphorylated HSP27 (A) and HSP27 phosphorylated at Ser82 (B) and Ser15 (C). Actin was used as a loading control and the relative levels of individual forms of HSP27 (HSP27, p-HSP27-Ser82 or p-HSP27-Ser15) after normalization were expressed as a percentage of the corresponding control level. Data represent the mean±S.E.M. of three separate experiments; *p<0.05 vs control.