Cardiac‐specific Overexpression of MiR‐133a in the Diabetic Heart Mitigates Mitochondrial Abnormality by Targeting TIM17A

Abstract

Abnormal mitochondria and reduced miR‐133a are hallmarks of diabetic mellitus (DM) hearts. However, it is unclear whether the lack of miR‐133a contributes to mitochondrial abnormalities in DM hearts. Decreased mitochondrial membrane potential is an indicator of abnormal mitochondria. Increased expression of TIM17A (translocase of the inner membrane 17A), a core subunit of the mitochondrial TIM23 protein import complex, decreases mitochondrial membrane potential. We tested the hypothesis that lack of miR‐133a in the DM heart increases TIM17A that in turn decreases mitochondrial membrane potential and promotes mitochondrial abnormalities. To test the hypothesis, we created a novel DM mice strain where miR‐133a is overexpressed only in the heart (Akita/miR‐133aTg), by crossbreeding Ins2+/− Akita (spontaneous, genetic, T1DM) male with cardiac‐specific miR‐133a transgenic (miR‐133aTg) female mice. To determine the effect of DM on TIM17A and mitochondrial abnormalities, we used Ins2+/− Akita and its sibling Ins2+/+ WT mice. We used fourteen‐week male WT, Akita, Akita/miR‐133aTg and miR‐133aTg mice, and measured the cardiac levels of TIM17A (by QPCR) and mitochondrial abnormalities (by electron microscopy) in the heart (left ventricle). To investigate the mechanism by which miR‐133a regulates TIM17A, we used HL1 cells (murine cardiomyocyte cell line), treated them with normal (5mM) or high (25mM) doses of D‐glucose (simulating normal or diabetic environment, respectively) for 24 hours. To evaluate the impact of miR‐133a overexpression on mitochondrial abnormality in hyperglycemic cardiomyocytes, we treated HL1 cells with HG and miR‐133a mimic (miR‐133a+HG) or scrambled miRNA (scm+HG) for 24 hours. To determine the specific effect of TIM17A on mitochondrial membrane potential, we treated HG cells with TIM17A siRNA, and measured mitochondrial membrane potential using JC1 and TMRE dyes. To investigate if miR‐133a directly targets TIM17A, we performed in silico analysis and miRNA‐mRNA electrophoretic mobility shift assay (EMSA). We found that DM increases the cardiac levels of TIM17A (2.97±0.86 fold of WT), which is decreased in Akita/miR‐133aTg hearts (1.14±0.23 fold of WT). In Akita, we found disorganized and fragmented mitochondrial cristae, which was almost normal in Akita/miR‐133aTg hearts. In HL1 cells, HG increased the protein levels of TIM17A (1.77±0.21 fold of NG) and miR‐133a mimic treatment decreased TIM17A levels (0.87±0.25 fold of NG). HG treatment decreased mitochondrial membrane potential in HL1 cells (0.69±0.08 fold of NG using JC1, and 0.63±0.07 fold of NG using TMRE) but miR‐133a mimic treatment blunted HG‐induced decrease in mitochondrial membrane potential (1.07±0.08 fold of NG using JC1, and 0.92±0.04 fold of NG using TMRE). Interestingly, TIM17A knockdown in HG cells rescued mitochondrial membrane potential (1.23±0.05 fold of NG using TMRE). In silico analyses showed TIM17A as a potential target of miR‐133a (seed binding sequence: UUAGAAGAUAAAGGACCAA) and miRNA‐mRNA EMSA revealed that miR‐133a directly targets TIM17A (EMSA binding: WT 1.32±0.14 and mutant 0.40±0.22 using 3′UTR probes). In conclusion, we demonstrated that lack of miR‐133a induces TIM17A that in turn decreases mitochondrial membrane potential and promotes mitochondrial abnormalities, which could be ameliorated by miR‐133a mimic treatment.Support or Funding InformationAmerican Heart Association Postdoctoral fellowship award 16POST30180003 and National Institute of Health grants HL113281 and HL16205This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.