Transmission Of ER Stress From Cardiomyocytes To Macrophages And The Role Played In CVB3 Infection Induced VMC

The endoplasmic reticulum (ER) is a membranous network mainly recognized as a protein-folding factory, responsible for the biosynthesis, folding, assembly and modification in all eukaryotic cells. The UPR is an adaptive survival pathway that becomes activated by accumulation of misfolded proteins in response to a variety of cellular insults that result in ER stress. However, if ER stress is prolonged, or is combined with additional insults, this pathway can lead to cell apoptosis. Endoplasmic reticulum (ER) stress is triggered by many physiological and pathophysiological conditions, including ischemia, anoxia, glucose starvation, the misglycosylation of glycoproteins, calcium deprivation from the ER lumen, the abnormal formation of disulfides bond can instigate features of ER stress. Abnormalities of the UPR have been implicated in many diverse diseases, including neurodegenerative diseases, diabetes, atherosclerosis and viral infection.Coxsackievirus B3 type (CVB3) is one of the important reasons for viral myocarditis (VMC). It belongs to the human enterovirus B cluster in the family picornaviridae and is closely associated with acute/chronic myocardial disease and dilated cardiomyopathy cases. In recent years, the incidence of VMC is increasing in our country and the pathologic mechanism remains unelucidated. More and more evidence showed that besides viral infection, cardiac injury was also associated with inflammatory infiltration in viral myocarditis. We have focused on macrophages because they consisted of the major cell type. There are papers suggest that ER stress was involved in regulation of macrophage function and influence the progression of disease. So, we want to know whether macrophage ER stress underlie the development of CVB3 induced VMC.In the present study,6-8 weeks male BALB/c mice were infected with CVB3 and lever of ER stress in cardiomyocytes and heart-infiltrating macrophages between the normal mice and mice with myocarditis was determined. Results showed that CVB3 infected mice cardiomyocytes and heart-infiltrating macrophages had significantly increased level of ER stress when compared to normal mice. Interestingly, when we stimulated RAW264.7 macrophages and bone marrow derived macrophages (BMDMs) with CVB3 in vitro, we could not detect ER stress in both of these two kinds of cells, but when we stimulate neonatal mice cardiomyocytes cultured in vitro or Hela cells, we could detected ER stress too. It is reported that ER stress in tumor cells can be transferred to bone marrow-derived myeloid cells by secrete a mediator(s). We suggest that ER stress in macrophages was transmitted from CVB3 infected cardiomyocytes. We established ER stress transmit system in vitro to confirm our hypothesis, and also explored the role of ER stress in macrophages during CVB3 infection both in vivo and in vitro. The present investigation is including the following aspects:1. The detection of ER stress in CVB3 infection induced VMC.We obtained and purified cardiomyocytes from normal and CVB3 infected mice and detected ER stress condition. We found that GRP78 and GRP94, the major indicator of ER stress was upregulated one day after infection, and peaked on 3 days after infection. Then we isolated heart-infiltrating macrophages by flow cytometer, and determined the ER stress response. We found that GRP78 and GRP94 expression gradually increased from 3 days after CVB3 infection. In a word, CVB3 infection can induce ER stress in cardiomyocytes and heart-infiltrating macrophages.2. CVB3 can not induce ER stress in macrophages but ER stress in cardomyocytes induced by CVB3 infection can transmit to macrophaesWe found that CVB3 infection can induce ER stress in Hela cells and neonatal mice cardiomyocytes, but when we want to verify this result in RAW264.7 cells and BMDMs we found that CVB3 can not induce ER stress in macrophages directly. We suggest that the ER stress we detected in heart-infiltrating macrophages was transmitted from cardiomyocytes. We cultured RAW264.7 cells with ER stress conditioned media from CVB3 infected cardiomyocytes, the results suggest that cardiomyocytes can secrete a mediator(s) that causes macrophages to initiate an ER stress response in macrophages and it was in dose-dependent manner.3. The role of transmissible ER stress in macrophages played in CVB3 infection induced VMC.From part 1 we know that CVB3 infection can induce ER stress in heart-infiltrating macrophages, and then we treated cells with ER stress inhibitor TUDCA (500μg/ml) in vitro, and examined the releasing of proinflammatory factors TNF-a and IL-6. The level of IL-6 was dramatically declined following the suppression of ER stress. Then we explored the role of ER stress in RAW264.7 cells, cells were cultured in two dishes and infected with CVB3 (MOI=10), one was stimulated with ER stress inducer tunicamycin (Tm) and the other one added same volume of DMSO, as expected, the releasing of IL-6 was increased along with the up-regulation of ER stress.Then we further investigate the affect of macrophages ER stress in the progression of VMC, we eliminated mice macrophages by tail vein injection of Lip-Cl2MDP and infected mice with CVB3, then transferred ER stress induced BMDMs and ER stress suppressed BMDMs into mice respectively. Asscessed the severity of VMC in mice by histology analysis of HE-stained heart sections, body weight loss, serum creatine kinase (CK) and CK-MB activities and cardiac systolic function test. The results showed that the susceptibility to viral myocarditis did increase when ER stress induced macrophages were transferred.Above all, it’s the first time to find that CVB3 infection can not induce ER stress in macrophages directly, and the ER stress in heart-infiltrating macrophages was transmitted from cardiomyocytes. And we also found that ER stress in macrophages played an important role during the progress of VMC induced by CVB3. So our study further clarified the pathogenesis of CVB3 induced viral myocarditis and provided a potential target for the treatment of viral myocarditis.