Mitochondrial proteome disruption in the diabetic heart through targeted epigenetic regulation at the mitochondrial heat shock protein 70 (mtHsp70) nuclear locus.

>99% of the mitochondrial proteome is nuclear-encoded. The mitochondrion relies on a coordinated multi-complex process for nuclear genome-encoded mitochondrial protein import. Mitochondrial heat shock protein 70 (mtHsp70) is a key component of this process and a central constituent of the protein import motor. Type 2 diabetes mellitus (T2DM) disrupts mitochondrial proteomic signature which is associated with decreased protein import efficiency. The goal of this study was to manipulate the mitochondrial protein import process through targeted restoration of mtHsp70, in an effort to restore proteomic signature and mitochondrial function in the T2DM heart. A novel line of cardiac-specific mtHsp70 transgenic mice on the db/db background were generated and cardiac mitochondrial subpopulations were isolated with proteomic evaluation and mitochondrial function assessed. MicroRNA and epigenetic regulation of the mtHsp70 gene during T2DM were also evaluated. MtHsp70 overexpression restored cardiac function and nuclear-encoded mitochondrial protein import, contributing to a beneficial impact on proteome signature and enhanced mitochondrial function during T2DM. Further, transcriptional repression at the mtHsp70 genomic locus through increased localization of H3K27me3 during T2DM insult was observed. Our results suggest that restoration of a key protein import constituent, mtHsp70, provides therapeutic benefit through attenuation of mitochondrial and contractile dysfunction in T2DM.

Exploring the mitochondrial microRNA import pathway through Polynucleotide Phosphorylase (PNPase).

Cardiovascular disease is the primary cause of mortality for individuals with type 2 diabetes mellitus. During the diabetic condition, cardiovascular dysfunction can be partially attributed to molecular changes in the tissue, including alterations in microRNA (miRNA) interactions. MiRNAs have been reported in the mitochondrion and their presence may influence cellular bioenergetics, creating decrements in functional capacity. In this study, we examined the roles of Argonaute 2 (Ago2), a protein associated with cytosolic and mitochondrial miRNAs, and Polynucleotide Phosphorylase (PNPase), a protein found in the inner membrane space of the mitochondrion, to determine their role in mitochondrial miRNA import. In cardiac tissue from human and mouse models of type 2 diabetes mellitus, Ago2 protein levels were unchanged while PNPase protein expression levels were increased; also, there was an increase in the association between both proteins in the diabetic state. MiRNA-378 was found to be significantly increased in db/db mice, leading to decrements in ATP6 levels and ATP synthase activity, which was also exhibited when overexpressing PNPase in HL-1 cardiomyocytes and in HL-1 cells with stable miRNA-378 overexpression (HL-1-378). To assess potential therapeutic interventions, flow cytometry evaluated the capacity for targeting miRNA-378 species in mitochondria through antimiR treatment, revealing miRNA-378 level-dependent inhibition. Our study establishes PNPase as a contributor to mitochondrial miRNA import through the transport of miRNA-378, which may regulate bioenergetics during type 2 diabetes mellitus. Further, our data provide evidence that manipulation of PNPase levels may enhance the delivery of antimiR therapeutics to mitochondria in physiological and pathological conditions.