Integrative mechanisms, Novel diagnostic and Therapeutic approachesP405Targeted deletion of Dicer alters the cardiac mitochondrial and myofilament subproteome

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Abstract

Background

Dicer is critical for the processing of double stranded RNAs, including microRNAs. In patients with dilated cardiomyopathy and heart failure, a reduction in the expression levels of Dicer is observed. Subsequent loss-of-function studies in mice resulted in a heart failure phenotype.

Methods and Results

To assess the effect of conditional Dicer gene deletion on the cardiac proteome, hearts from double-transgenic tamoxifen-treated MHC-MCM/DicerF/F mice and control tamoxifen-treated DicerF/F mice were subfractionated and analyzed by proteomics using a high-mass accuracy tandem mass spectrometer (LTQ Orbitrap XL). KEGG pathway and protein interaction network analyses of the top 150 differentially expressed protein revealed that most changes after targeted Dicer deletion in cardiomyocytes were confined to mitochondria, in particular protein complexes of the oxidative phosphorylation chain. This is consistent with previous observations that mitochondria isolated from Dicer depleted cardiomyocytes were primarily uncoupled and showed a reduction in energy dependent pore openings. The mitochondrial changes coincided with an upregulation of sorcin, a ryanodyne receptor modulator that regulates the contractile state of the myocardium by affecting intracellular calcium. In addition, our proteomic study indicated degradation of myofibril proteins, such as titin, as well as changes in other Z-disc or titin-interacting proteins (four and a half LIM domains protein 2, LIM domain-binding protein 3, myozenin2, etc.) known to be central to myofilament regulation. Z-disks in Dicer-depleted hearts showed abnormal morphology associated with frequent disruption in sarcomere structures. These alterations were accompanied by an increase of matrix metalloproteinase 9 (MMP-9) in Dicer depleted hearts, which can mediate contractile dysfunction of cardiomyocytes.

Conclusions

Changes in cardiac mitochondria and myofibrils are among the most prominent changes in the cardiac proteome after targeted Dicer depletion providing an explanation for the pronounced cardiac disease phenotype. This proteomic approach opens up new avenues for cardiac microRNA research.

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