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

Abstract

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.

Keywords: Bioenergetics; Diabetes mellitus; Mitochondria; microRNA.

Fig. 1

Ago2 expression levels in human and mouse models of type 2 diabetes mellitus. Ago2 expression from non-diabetic (closed bars, n = 5) and diabetic (open bars, n = 5) human atrial tissue in (A) SSM and (B) IFM subpopulations. Ago2 expression levels in non-diabetic and db/db mice in (D) SSM and (E) IFM subpopulations. Ago2 expression levels in cytoplasmic fractions of both (C) human and (F) mouse cardiac tissue. Western blotting quantified to COXIV in the mitochondria. Values are means ± SE. † = P ≤ 0.075. Ago2 = Argonaute 2, SSM = subsarcolemmal mitochondria, IFM = interfibrillar mitochondria, ND = non-diabetic, T2DM = type 2 diabetes mellitus.

Fig. 2

PNPase expression levels in human and mouse models of type 2 diabetes mellitus. PNPase expression from non-diabetic (closed bars, n = 5) and diabetic (open bars, n = 5) human atrial tissue in (A) SSM (P ≤ 0.055) and (B) IFM subpopulations. PNPase expression levels in non-diabetic and db/db mice in (C) SSM and (D) IFM subpopulations. PNPase expression levels in cytoplasmic fractions of both (E) human and (F) mouse cardiac tissue. Western blotting quantified to COXIV in the mitochondria. Values are means ± SE. * = P ≤ 0.05, † = P ≤ 0.075. PNPase = Polynucleotide Phosphorylase, SSM = subsarcolemmal mitochondria, IFM = interfibrillar mitochondria, ND = non-diabetic, T2DM = type 2 diabetes mellitus.

Fig. 3

Evaluation of the binding of Ago2 and PNPase through immunoprecipitation in db/db and littermate control mice. Ago2 was immunoprecipitated then transferred and run on a SDS-PAGE gel where the (A) SSM and (B) IFM were probed for PNPase, in both the non-diabetic (closed bars, n = 5) and diabetic (open bars, n = 4) groups. Alternatively, PNPase was immunoprecipitated then transferred and run on a SDS-PAGE gel where the (C) SSM and (D) IFM were probed for Ago2. Values are means ± SE. * = P ≤ 0.05, † = P ≤ 0.075. Ago2 = Argonaute 2, PNPase = Polynucleotide Phosphorylase, SSM = subsarcolemmal mitochondria, IFM = interfibrillar mitochondria, ND = non-diabetic, input = 50% input control.

Fig. 4

The relationship between miRNA-378 and bioenergetics. (A) MiRNA-378 levels in the SSM and IFM, in both the non-diabetic (closed bars, n = 4) and db/db (open bars, n = 4) groups. (B) ATP6 levels in the SSM and IFM. (C) ATP Synthase activity of mitochondria from control and db/db animals. qPCR C_t values were normalized to U6 expression. Western blotting quantified to COXIV. Values are means ± SE. * = P ≤ 0.05. SSM = subsarcolemmal mitochondria, IFM = interfibrillar mitochondria, ND = non-diabetic.

Fig. 5

The influence of PNPase on mitochondrial miRNA-378 in cell culture models. (A) HL-1 and HL-1-378 miRNA-378 fold change. (B) ATP synthase activity of HL-1 and HL-1-378 cell lines. (C) Representative western blot of transient PNPase overexpression and knockdown in HL-1 cells. (D) Expression of mitochondrial miRNA-378 in HL-1 cells during normal, overexpressing, and knocked down expression of PNPase. (E) Expression of mitochondrial miRNA-378 in HL-1-378 cells during normal, overexpressing, and knocked down expression of PNPase. qPCR C_t values were normalized to U6 expression. N = 4 for each group. Values are ± SE. * = P ≤ 0.05. HL-1 PNPase = murine cardiomyocyte cell line transiently overexpressing or knocking down Polynucleotide Phosphorylase (PNPase), HL-1-378 PNPase = murine cardiomyocyte cell line stably overexpressing miRNA-378a-3p while transiently overexpressing or knocking down Polynucleotide Phosphorylase (PNPase).

Fig. 6

The influence of PNPase and mitochondrial miRNA expression on bioenergetics. ATP synthase activity was measured in (A) HL-1, (B) HL-1-378, and (C) HL-1-378 with antimiR-378 cells with manipulations to the expression of PNPase. (D) Western blot analysis for mitochondrial ATP6 in HL-1-378 control, PNPase overexpressing, and PNPase knockdown cells. (E) Western blot analysis for mitochondrial ATP6 in HL-1-378 cells transfected with antimiR-378 with normal and overexpressing levels of PNPase. Western blotting quantified to COXIV in the mitochondria. Values are means ± SE. * = P ≤ 0.05. HL-1 PNPase = murine cardiomyocyte cell line transiently overexpressing or knocking down Polynucleotide Phosphorylase (PNPase), HL-1-378 PNPase = murine cardiomyocyte cell line stably overexpressing miRNA-378a-3p while transiently overexpressing or knocking down Polynucleotide Phosphorylase (PNPase). AntimiR PNPase = HL-1-378 cell line transfected with antimiR-378 while transiently overexpressing or knocking down Polynucleotide Phosphorylase (PNPase).

Fig. 7

MiRNA-378 antimiR import into the mitochondria visualized through fluorescence imaging and flow cytometry. Cells were transfected with (A) miRNA-378 antimiR LNA or scramble LNA in both HL-1 and HL-1-378 cell lines and visualized qualitatively for transfection efficiency. (B) Flow cytometry was used to measure mean fluorescence of mitochondria, gating to Mitotracker Deep Red 633. (C) Samples were compared to respective no-stain GFP controls (Mean fluorescence ≤ 0.25). Control HL-1 and HL-1-378 cells, (n = 4) and PNPase overexpression and knockdown in each cell line (n = 4) were evaluated. (D) Observed changes during flow cytometric analysis. Values are means ± SE. * = P ≤ 0.05. LNA = locked nucleic acid for miRNA-378 antimiR LNA, scram = locked nucleic acid for Negative Control A, HL-1 PNPase = murine cardiomyocyte cell line transiently overexpressing or knocking down Polynucleotide Phosphorylase (PNPase), HL-1-378 PNPase = murine cardiomyocyte cell line stably overexpressing miRNA-378a-3p while transiently overexpressing or knocking down Polynucleotide Phosphorylase (PNPase).

Fig. 8

Graphical illustration of PNPase and the changing dynamics in the mitochondrion during type 2 diabetes mellitus. In the non-diabetic state, fewer PNPase proteins are located within the intermembrane space of the mitochondria, resulting in fewer miRNA-378 transcripts being imported. During type 2 diabetes mellitus, an increase in PNPase expression results in increased associations with Ago2, which provide for additional influx of miRNA-378 into the mitochondrion. These miRNA-378 transcripts target mt-ATP6 mRNA, resulting in fewer catalytically active proteins. The lack of mt-ATP6 protein expression contributes to a decrease in bioenergetics through disrupting the F₀ complex of ATP synthase. Ago2 = Argonaute 2, mt-ATP6 = mitochondrial ATP synthase F₀ subunit 6, PNPase = Polynucleotide Phosphorylase.