Although exercise mitigates diabetic complications and myopathy, the mechanism is unclear. The long-term goal of this project is to understand the mechanism of diabetic myopathy. Studies from previous funding period revealed causative role of endothelial-myocyte (E-M) uncoupling in cardiomyopathy. The connexin -37 and -43 are associated with endothelial and myocyte coupling; and mitochondria contains connexin-43. Matrix metalloproteinase-9 (MMP-9) disrupts connexins and is activated in diabetes, unequivocally. Interestingly, the exercise decreases MMP-9 activity. Exosomes, 50-100 nm, nanovesicles, contain mir-RNAs and are released from myocytes. In this renewal application we will determine the role of exercise-mediated release in exosome containing mir-RNAs that mitigates the endothelium-myocyte, myocyte-myocyte and mitochondrial-myocyte uncoupling by down regulating the MMP-9. The central hypothesis of this proposal is that MMP-9 degrades connexins and causes endothelial-myocyte, myocyte-myocyte and mitochondria-myocyte uncoupling in diabetes. Exercise releases exosomes containing mir-RNAs that decreases MMP-9 and mitigates uncoupling. We will test the central hypothesis by following three specific aims.
Specific aim #1 : To determine whether the exercise releases exosomes that contain mir-29b, mir-323, mir-455 and mir-466 which negatively regulate MMP-9 and mitigate fibrosis and uncoupling.
Specific aim #2 : To determine whether endothelial-myocyte uncoupling is caused by perivascular/pericapillary fibrosis and myocyte-myocyte uncoupling is caused by interstitial fibrosis in diabetes and exercise mitigates these conditions.
Specific aim #3 : To determine whether the dys-synchronization of mitochondrial fusion-fission causes mitochondria-myocyte uncoupling (mitochondrial remodeling) in diabetes and exercise alleviate. To evaluate the release of exosomes and their effect on MMP-9 and consequently Cx-37 and Cx-43, adult male db/db and controls db/+, wild type (WT-C57 and FVB) mice, MMP-9 KO, db/db/MMP-9 DKO, Cx-37-Tg, db/db/Cx-37-Tg; Cx-43-Tg, and db/db/Cx-43-Tg mice with and without exercise will be used. Plasma and cardiac tissue exosomes will be isolated by ultracentrifugation/exoquick and characterized by electron microscopy/FACS/MACS. The levels of mir466, mir455, mir29b, mir323 will be determined by RT-PCR and in situ hybridization. These studies will determine the mechanism of muscular structural, functional and mitochondrial remodeling and exosomal therapeutic ramifications for diabetic myopathy.
Because cardiac matrix is unique, general matrix metalloproteinase-9 (MMP-9) inhibitors may not mitigate the cardiac remodeling (Clinical trial- PREMIER-Pevention of Myocardial Infarction Early Remodeling, using the MMP-9 inhibitor PG-116800). Exosomes that carry microRNAs can provide alternate treatment strategy to inhibit MMP-9 since exosomes are non-immunogenic and have no side effects. Myocyte releases exosomes and exosomes contains miRNA. The miRNA inhibits MMP- 9 by binding to the 3'UTR region. These studies will determine the mechanism of muscular structural, functional and mitochondrial remodeling and exosomal therapeutic ramifications for diabetic myopathy.
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