Characterization of opioid receptors in post-heart transplantation, diabetic heart, great vessels, and cardioprotective role in myocardial ischemia-reperfusion injury

Background: Despite several functions of opioid receptors/ORs in cardiovascular physiology and neurotransmission, expression in post-heart transplantation, diabetic heart, great vessels, and cardioprotective roles in neuropathic models of diabetic rat heart has not yet been addressed. The aims of this study were i) to investigate whether myocardial opioidergic system in transplanted and diabetic heart was altered, ii) to study the modulation of some pro-survival signaling proteins upon ORs blockade in diabetic rat model of post-myocardial ischemia-reperfusion injury (IRI), iii) to measure levels of apoptotic nuclei and infarct size in the presence of naloxone, iv) to evaluate and compare the expression of kappa (κ) and delta (δ) ORs in aorta and pulmonary arteries in rats. Materials and methods: Endomyocardial biopsy from the orthotopically transplanted heart (OTH, human); tissues from heterotopic transplanted heart (HTH), diabetic rat heart in the presence and absence of naloxone after ischemia-reperfusion/IR following body weight and blood glucose measurement were collected and preserved in formalin and liquid nitrogen. The tissues were processed for total-RNA isolation, protein extraction, immunohistochemistry (IHC), immunofluorescence (IF), TUNEL, and Hematoxylin & eosin (H&E). The optical density (OD) of ORs immunoreactivity and a neuronal marker (CGRP-1) were measured. The pro-survival signaling pathways involved in alterations, phosphorylation levels of GSK-3α/β and p38, and TUNEL positive apoptotic nuclei were evaluated after IR-induction. 2, 3, 5-triphenyl tetrazolium chloride/TTC staining was also applied to measure levels of infarction of IR-induced rat heart with and without naloxone. H&E staining was performed to: grade the level of rejection; evaluate histopathology of sciatic nerve and pancreas in diabetic rats and any concurrent structural abnormalities of heart tissue. Immunohistochemically, κ- and δ-ORs were evaluated in endothelial and vascular smooth muscle cells in the aorta and pulmonary arteries of rats. Results: IHC and IF observations showed the expression of δ- & κ-ORs in the heart with a significant reduction in the OD of DOR-1 & KOR-1 immunoreactivity in post-heart transplantation and in Streptozotocin-induced diabetic rat heart. However, MOR-1 was not detected in the rat heart. Dual labeling signals of KOR-1 with DOR-1 co-expression were observed in both hearts of human and rats. The reduced OD of DOR-1 immuno-positive myocytes significantly correlated with the reduction of neuronal marker (CGRP-1) immunoreactivity. Messenger RNA transcripts encoding the Oprd1 and Oprk1 were detected in human and rats, and downregulated in transplanted and diabetic hearts. Nevertheless, Oprm1 was not identified in rats` heart. Undetected Oprm1 RT-qPCR products also were shown by agarose gel electrophoresis. Densitometric quantification of immunoblots revealed that DOR-1 and KOR-1 proteins were also downregulated in transplanted and diabetic hearts of rats. Furthermore, an elevation of apoptotic nuclei of myocytes in transplanted and diabetic heart was observed. Histopathological examination of transplanted heart tissue demonstrated lymphocytes infiltrate in orthotopically and heterotpic transplanted heart in human and rats, respectively. Moreover, lower body weight and fasting blood glucose levels and abnormal distribution and shrinkages of βeta-cells of islet of pancreas, and connective tissue fibrosis around the epineurium and axonal swelling of the sciatic nerve were detected in diabetic rats. Mild morphological distortion of myocytes was observed in diabetic heart in the presence and absence of naloxone. Process of cell-signaling showed that blockade of ORs by naloxone reduces the levels of phosphorylated pro-survival kinases (AKT, ERK1/2) in diabetic and IR-induced rat heart. On the contrary, the extent of phosphorylation of GSK-3β significantly elevated in the presence of naloxone. On the other hand, absence or poor phosphorylation of GSK-3α was observed in almost all groups tested. Moreover, elevation of p38 phosphorylation, TUNEL positive nuclei, and fibrosis was observed in the presence of naloxone after IR. TTC-stained ventricular slices showed a higher percentage of infarct size after ORs blockade. Comparative study of KOR-1 and DOR-1 on the aorta and pulmonary artery showed presence of κ- and δ-ORs in endothelial and vascular smooth muscle cells in the aorta and pulmonary arteries in rats dominantly KOR. Conclusion: The δ- & κ-ORs possess a protective role in the heart against ischemia-reperfusion injury; however, the down-regulation of ORs may compromise the effectiveness of pharmacological activities of opioids in transplanted and diabetic hearts that could reduce the potential role of ORs in the regulation of cardiac tissue. The ORs might promote cell survival by mediating the action of Akt and ERK1/2. The phosphorylation of GSK-3β and p38 might also be involved in ORs in regulation of myocardial tissue. The ORs dominantly KOR also play role in endothelial and smooth muscle cells in vascular system in rats. Keywords: Opioid receptors, Cardiac transplantation, Cellular rejection, Ischemia-reperfusion injury, Diabetic neuropathy, Apoptosis, Myocardial infarction, Endomyocardial biopsy, Denervation, Vascular System