Subunit of mitochondria and hypertension control via miRNA

Abstract

Background: Excessive reactive oxygen species (ROS) generated in the mitochondria is known to be a causal event in hypertensive cardiomyopathy. Multiple recent studies suggest that nuclear genome-encoded miRNAs are able to translocate to the mitochondria to modulate mitochondrial activities, but the medical significance of such new miRNA function has remained unclear. Methods: Expression levels of representative proteins of mitochondrial subunits and ROS production were detected in hearts from Wistar rats and spontaneous hypertensive rats (SHRs) by Western blotting and dihydroethidium (DHE) staining, respectively. By using siRNAs, we identified a critical role of mitochondrial cytochrome b (mt-Cytb) in ROS generation. Next, mt-Cytb was predicted as target of miRNA-21 (miR-21) by bioinformatic analysis, followed by validations using real-time PCR and Ago2 immunoprecipitation. Furthermore, the mechanisms underlying miR-21 enhanced mitochondrial mt-Cytb translation were verified by polysome analysis. To distinguish the mitochondrial ROS from total ROS in cultured cells, MitoSOX™ Red and 2,7-Dichlorodihydrofluorescein diacetate staining were performed in H9c2 cells. In addition, circulating miR-21 levels were determined in 100 hypertensive patients and 120 controls. Finally, SHRs were treated with rAAV-miR-21 via tail vein, followed by blood pressure monitoring with a photoelectric tail-cuff system. Cardiac structure and functions were assessed by echocardiography and catheter manometer system. Moreover, ROS production in various organs was determined by DHE staining. Results: We observed a marked reduction of mt-Cytb in the heart of SHRs. Down-regulation of mt-Cytb by siRNA recaptured some key disease features, including elevated ROS production. Through computational prediction, Ago2 immunoprecipitation and polysome analysis, we found that miR-21, which was induced in SHRs and we showed as part of the compensatory program, directly targeted mt-Cytb, leading to enhanced translation in transfected H9c2 cells. Circulating miR-21 levels in 100 hypertensive patients were significantly higher than those in 120 controls, showing positive correlation with blood pressure. Remarkably, rAAV-mediated delivery of miR-21 was sufficient to reduce blood pressure and attenuate cardiac hypertrophy in SHRs. Conclusions: Our findings reveal a positive function of miR-21 in mitochondrial translation, which is sufficient to reduce blood pressure and alleviate cardiac hypertrophy in SHRs. This observation suggests a novel theoretical ground for developing miRNA-based therapeutics against hypertension.