In the present study, we demonstrated an important action of ticagrelor, a drug directly targeting P2Y12-receptors, on cardiomyocytes-derived exosomes in hyperglycemic cardiomyocytes. Our present data further emphasized that ticagrelor exerts its regulatory effect on diabetic cardiomyopathy through exosomal modulation beyond its antiplatelet action. We demonstrated the importance of ticagrelor-pretreatment of cardiomyocytes to obtain the benefits of exosome modulation in heart functions under pathological conditions including diabetes in vitro.
Among long-term complications of DM, there are both macro-level and micro-level abnormalities, leading to heart diseases besides other complications. DM, at cellular levels, is associated with detectable changes in intracellular function and structure of cardiomyocytes through effecting several molecular mechanisms including oxidative stress, inflammation, apoptosis and autophagy, miRNA regulation, mitochondrial dysfunction, and ER stress 19–21. Since DM is a major killer worldwide and its very rapid rise emphasizes a serious threat to humans, there are great efforts to develop novel therapeutic approaches for DM management 16, 17, 22.
DM patients have a high risk for cardiovascular disease, named diabetic cardiomyopathy. Diabetic cardiomyopathy, being a noticeably different pathology from heart failure, was first defined by Rubler and co-workers 23, which does not include coronary atherosclerosis but is characterized by an irreversible loss of cardiomyocytes, the contractile cellular units of the heart. Over the last two decades, studies focused on stem cell therapy as a potential approach for cardiac repair 24 and it is observed that a large part of the benefit of the injection of stem and progenitor cells into injured hearts is mediated by secreted factors 25. The study by Wang et al. 26 advances our understanding of the role of cardiomyocyte exosomes and hints at the complexity of exosomal cargo under diabetic conditions. Here, for the first time in the literature, we have explored the role of ticagrelor-pretreatment of H9c2 myocardium cells, and then on the degree of beneficial effects of exosomes derived from them.
Although it was reported that ticagrelor administration blockades tumor angiogenesis via suppressing inflammation 27,11, the increased co-localization of c-Kit protein, a marker of cardiac stem cells and CD31 protein, a marker of endothelial cells, around blood vessels upon ticagrelor treatment demonstrated the positive effect of ticagrelor on angiogenesis following myocardial ischemia-reperfusion in the rat 28. Moreover, the increased levels of circulating endothelial progenitor cells in diabetic patients after ticagrelor administration were also reported 29. Since the angiogenic contribution of the ticagrelor is known, it is conceivable that ticagrelor may exert this effect via modulating the angiogenic properties of exosomes derived from ticagrelor-treated cardiomyocytes. We found- for the first time- that ticagrelor-pretreatment markedly induces branche, mesh, segment, and node formation of HUVECs at the 6th and 24th hours of incubation. Endothelial cells are more susceptible to hyperglycemia-induced damage than other cell types 30. Diabetes causes endothelial dysfunction through the impairment of several mechanisms, including oxidative stress, inflammatory activation and increased permeability of the endothelial cell layer in the myocardium, and decreased capillary density 31,32. Hyperglycemia-mediated activation of the diacylglycerol (DAG)-PKC signaling pathway is involved in the induction of oxidative stress excessive production of free radicals, which in turn promote endothelial dysfunction which leads to diabetic cardiomyopathy 31,33. Through the above results, endothelial cells may be considered as a potential therapeutic target in the treatment of diabetes. Here, we have also demonstrated that exosomes released from H9c2 cells pretreated with 1-µM ticagrelor significantly increase endothelial cell migration. Considering that under hyperglycemic conditions, endothelial cell viability and migration reduces, our findings could provide that ticagrelor-pretreatment reverses endothelial dysfunction in an exosome-mediated paracrine manner.
It is well established that ticagrelor therapy administered in diabetic patients demonstrated a direct beneficial effect against heart dysfunction beyond its antiplatelet action 34,35. Olgar et al reported that ticagrelor provided a significant improvement in mitochondrial membrane potential, a marked decrease in cellular ROS production, a normalization of the increased resting level of cytosolic Ca2+, and preservation of cellular ATP production. Moreover, in that study, it was also demonstrated that ticagrelor alleviates enhanced ER stress, autophagosomes formation, and apoptosis by inhibiting the expression of the genes involved in these molecular pathways 18. However, the exact subcellular mechanisms by which ticagrelor exerts its cardioprotective, anti-hypoxic, and anti-apoptotic effect remains to be elucidated. Consistently, Casiere and co-workers have shown that long-term treatment with the lower dose of ticagrelor enhances anti-hypoxic and anti-apoptotic effects of exosomes released from human cardiac-derived mesenchymal progenitor cells (hCPC) via attenuating the increase of HIF1α levels in hypoxic cardiomyocytes 16. This finding revealed the new role of ticagrelor in the regulation of anti-apoptotic and anti-hypoxic properties of exosomes derived from hCPCs, which are stem/progenitor cells resident in the adult heart 36. Taken into consideration the well-established actions of ticagrelor, particularly, associated with exosomal modulation in terms of cardiac regeneration and cardiomyocyte death, in this study, we have demonstrated that ticagrelor induced H9c2 exosomes have a direct beneficial effect on hyperglycemic ventricular cardiomyocyte causing a marked decrease in cellular ROS production.
Superoxide production is also associated with ENT1, while ENT1 plays a central role in the determination of the extracellular content of adenosine, which is generated extracellularly by the degradation of ATP 37. Upon ischemic reperfusion, ENT1 directly involves the formation of superoxide radicals 38 and plays an essential role in cardioprotection via modulating purine nucleoside-dependent signaling in the heart 18,39. Taken together, our data revealed that ticagrelor exerts its cardioprotective effect on oxidative stress-associated several factors through exosomal modulation.
Diabetic cardiomyopathy can be a major cause of diabetes-related mortality. Emerging preclinical and clinical evidence demonstrates that diabetes induces cardiomyocyte apoptosis. On the other hand, cardiac autophagy is still a controversial issue. In addition to the beneficial effect of autophagy during ischemic reperfusion 40, uncontrolled excessive induction of autophagy may contribute to autophagic cardiomyocyte death 41. Bcl-2 family members Bnip3 and Beclin are key regulators of necrosis, autophagy, and/or apoptosis 42. Consistent with the previous study 18 our data demonstrated treatment of HG cardiomyocytes with exosomes derived from ticagrelor included H9c2 cells causes the inhibition of the expression of autophagy and apoptosis markers, which are drastically upregulated depending on the hyperglycemia. Of note, the control-exosome treatment has shown a remarkable effect in induced autophagy formation and apoptosis in HG cardiomyocytes.
Furthermore, it is also well accepted that autophagy was induced as an adaptive response against ER stress since it was sensitive to ER-stress inhibition 43. Supporting these statements, it was reported that ticagrelor can directly affect cardiomyocytes and provide marked protection against ER stress and dramatic induction of autophagosomes, and therefore, can alleviate the ER stress-induced increases in oxidative stress and cell apoptosis during insulin resistance 18,44. In the present study, we have shown that ticagrelor exerts this suppressive effect on markedly increased ER stress via modulating released exosomes from H9c2 cells which cause the significant downregulation of mRNA level of ER stress markers. Finally, we have also tested have also evaluated the changes in mRNA levels of oxidative stress and cardiomyopathy-related associated cellular and exosomal miRNAs. Previously, lipopolysaccharide-treated rat cardiomyocytes showed a marked decrease in expression of miR-499 that inhibited the expression of pro-apoptotic genes and upregulated expression of the anti-apoptotic gene BCL-xL by targeting SOX6 and PDCD4, which play important roles in cardiac differentiation of P19CL6 cells 45 and the protection of cardiomyocytes against H2O2-induced injury 46, respectively. In a mouse diabetic cardiomyopathy model, Chen et al demonstrated that miR-133a, expressed in both skeletal and cardiac muscle, significantly decreased the expression of TGF-β1 signaling and increased the expression of acidic fibroblast growth factor1 (FGF1) that could induce ERK1/2 phosphorylation accompanied by enhanced production of extracellular matrix proteins, FN1 and COL4A1, hallmarks of cardiac fibrosis 47. miR-133b, which is expressed specifically in skeletal muscle, was reported to be downregulated in STZ-induced diabetic rats concerning increased oxidative stress 21 and protected H9c2 against hypoxia injury via downregulation of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) 48. Downregulated expression of cellular miR-499, miR-133a, and miR-133b in HG-cardiomyocytes associated with diabetes was drastically upregulated upon ticagrelor-induced H9c2 exosome treatment. Considering the important fact, a diabetic heart composes both healthy and diabetic cardiomyocytes, we have also examined the therapeutic paracrine effect of DM cardiomyocytes following the ticagrelor treatment. Exosomal miR-499, miR-133a, and miR-133b in hyperglycemic cardiomyocytes were upregulated upon ticagrelor treatment. These results supported that ticagrelor treatment may promote the therapeutic effect of exosomes released from both healthy and hyperglycemic cardiomyocytes. In the current study, we have also examined predicted targets of miR-499, miR-133a, and miR-133b linked with biological processes. biological and molecular functions, which are targets of miR-499, miR-133a, and miR-133b. These miRNAs have been involved as negative regulators in the pathway of apoptosis, positive regulators of cell migration, and wound healing, and cellular stress response. Such biological relevance of target genes elucidated through GO-enrichment may account for the pathogenesis in DCM through their involvement especially in heart development (GO:0007507) and positive regulation of glucose metabolic process (GO:0010907) (p < 0.05). Taken together, this analysis supports the effect of ticagrelor pretreatment on suppression of cellular stress, ROS production, cell death in addition to the contribution of angiogenesis and cell migration via modulation of exosomal profile.