A major cause of diabetes is impairment of glucose uptake by skeletal muscle that causes pumping of extra glucose into blood leading to hyperglycemia. Clinical studies revealed that diabetes causes cardiomyopathy and the chances of heart failure increases if the patient has diabetes. One of the mechanisms of cardiac dysfunction associated with diabetes is oxidative stress that activates latent matrix metalloproteinase-9 (MMP9), which in turn induces fibrosis and contractile dysfunction. However, the specific mechanisms for how oxidative stress activates MMP9, which leads to contractile dysfunction, have not been investigated. Our preliminary studies on HL1 cardiomyocytes suggest that inhibition of miR-133 induces MMP9 and over expression of miR-133 inhibits MMP9. The luciferase reporter assay revealed that miR-133 targets MMP9. Interestingly, glucose mediated induction of MMP9 is abrogated by miR-133. In the heart of diabetic Akita mice, myosin enhancer factor 2c (Mef2c- an inducer of miR-133) is alleviated, miR-133 is down regulated and MMP9 is robust. These results lead us to hypothesize that oxidative stress inhibits Mef2c causing attenuation of miR-133 that induces MMP9 leading to contractile dysfunction in diabetes. To address the hypothesis, we formulated three specific aims:
Aim#1 : To determine whether the miR-133 directly or indirectly inhibits the activation of MMP9. Hypothesis: MiR-133 directly inhibits MMP9 by targeting its 3/ UTR. It also indirectly inhibits MMP9 by inducing miR-466 and abrogating miR-705.
Aim# 2: To determine whether the oxidative stress inhibits Mef2c causing attenuation of miR-133 in diabetes. Hypothesis: The oxidative stress inhibits Mef2c that causes attenuation of miR-133 in diabetes.
Aim # 3: To determine whether the over-expression of miR-133 or Mef2c will inhibit MMP9 that in turn improve glucose uptake in skeletal muscle and ameliorates contractile dysfunction in diabetes. Hypothesis: The over expression of miR-133 and Mef2c inhibits MMP9 that enhances glucose uptake by skeletal muscle and mitigates contractile dysfunction of cardiomyocytes in diabetes. Our proposal unravels a new mechanism of regulation of MMP9 by miR-133. It also provides a new concept that miRNA inhibits a gene not only by directly targeting it rather it also induces / inhibits other miRNAs that indirectly influences the target gene. At translational level, the proposal will provide concrete evidence that over expression of miR-133 or ablation of MMP9 can ameliorate diabetic cardiomyopathy.

Public Health Relevance

Diabetes is a leading cause of morbidity and mortality and is rapidly increasing across the world posing great challenge to the current therapy. MicroRNAs (miRNAs) are tiny regulatory molecules that are recently emerged as a biomarker and therapeutic target for cardiovascular diseases and diabetic cardiomyopathy. Mir-133 is one of the major miRNAs established in cardiac hypertrophy. On the other hand, matrix metalloproteinase-9 (MMP9) is an endopeptidase that plays pivotal role in pathological cardiac remodeling and is robust in diabetic heart. However, the cross talk between miR-133 and MMP9 is unknown. Our preliminary studies suggest that miR-133 targets MMP9. In diabetes miR-133 is attenuated and MMP9 is induced. We tested whether up regulation of miR-133 can ameliorate MMP9 mediated cardiac dysfunction in diabetes. The preliminary results suggest that miR-133 inhibits MMP9 in diabetic cardiomyocytes. Also, ablation of MMP9 mitigates contractile dysfunction in diabetic Akita mice. These findings suggest that abrogation of MMP9 improves cardiac function in diabetes. The transcription of miR-133 is regulated by a nuclear factor-myosin enhancer factor 2c (Mef2c), which is down regulated in the diabetic heart. We proposed that in diabetes, oxidative stress alleviates Mef2c that in turn attenuates miR-133. The down regulation of miR-133 induces MMP9 causing contractile dysfunction leading to heart failure. The major goals of this project is to understand: a) the mechanism of miR-133 mediated regulation of MMP9, b) the effect of oxidative stress on Mef2c and miR-133 and c) whether over-expression of miR-133 or Mef2c and / or inhibition of MMP9 will ameliorate cardiac dysfunction in diabetes.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Special Emphasis Panel (ZRG1-MOSS-C (90))
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Wang, Lan-Hsiang
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University of Nebraska Medical Center
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Chavali, Vishalakshi; Tyagi, Suresh C; Mishra, Paras Kumar (2014) Differential expression of dicer, miRNAs, and inflammatory markers in diabetic Ins2+/- Akita hearts. Cell Biochem Biophys 68:25-35
Chavali, Vishalakshi; Nandi, Shyam Sundar; Singh, Shree Ram et al. (2014) Generating double knockout mice to model genetic intervention for diabetic cardiomyopathy in humans. Methods Mol Biol 1194:385-400
Mishra, Paras Kumar; Kuypers, Nicholas John; Singh, Shree Ram et al. (2013) Cardiac stem cell niche, MMP9, and culture and differentiation of embryonic stem cells. Methods Mol Biol 1035:153-63