Translational Regulation of the Mitochondrial Genome Following Redistribution of Mitochondrial MicroRNA in the Diabetic Heart

Abstract

Background: Cardiomyocytes are rich in mitochondria which are situated in spatially distinct subcellular regions, including those under the plasma membrane, subsarcolemmal mitochondria, and those between the myofibrils, interfibrillar mitochondria. We previously observed subpopulation-specific differences in mitochondrial proteomes following diabetic insult. The objective of this study was to determine whether mitochondrial genome-encoded proteins are regulated by microRNAs inside the mitochondrion and whether subcellular spatial location or diabetes mellitus influences the dynamics.

Methods and results: Using microarray technology coupled with cross-linking immunoprecipitation and next generation sequencing, we identified a pool of mitochondrial microRNAs, termed mitomiRs, that are redistributed in spatially distinct mitochondrial subpopulations in an inverse manner following diabetic insult. Redistributed mitomiRs displayed distinct interactions with the mitochondrial genome requiring specific stoichiometric associations with RNA-induced silencing complex constituents argonaute-2 (Ago2) and fragile X mental retardation-related protein 1 (FXR1) for translational regulation. In the presence of Ago2 and FXR1, redistribution of mitomiR-378 to the interfibrillar mitochondria following diabetic insult led to downregulation of mitochondrially encoded F0 component ATP6. Next generation sequencing analyses identified specific transcriptome and mitomiR sequences associated with ATP6 regulation. Overexpression of mitomiR-378 in HL-1 cells resulted in its accumulation in the mitochondrion and downregulation of functional ATP6 protein, whereas antagomir blockade restored functional ATP6 protein and cardiac pump function.

Conclusions: We propose mitomiRs can translationally regulate mitochondrially encoded proteins in spatially distinct mitochondrial subpopulations during diabetes mellitus. The results reveal the requirement of RNA-induced silencing complex constituents in the mitochondrion for functional mitomiR translational regulation and provide a connecting link between diabetic insult and ATP synthase function.

Keywords: cardiac; diabetes mellitus; genome; microRNAs; mitochondria; mitochondrial; myocytes.

Figure 1

Quality control assessment. (A) Gel electrophoresis of RNA isolated from whole heart and RNA from mitochondrial subpopulations. The lowest migrating green band is an internal standard. (B) Electropherograms of total RNA from whole heart and from isolated mitochondria. Scaling of the y-axis is done automatically, relative to the strongest signal within a single run. (C) Western blot analyses for GAPDH, calnexin, and ATPB protein content in cytosol, crude mitochondria, and percoll gradient purified mitochondria.

Figure 2

Relative normalized mitomiR expression patterns in cardiac mitochondrial subpopulations. (A) Hierarchical clustering heat map for the microarray analysis of mitomiR expression profiles in cardiac mitochondrial subpopulations. CS = control SSM, DS = diabetic SSM, CI = control IFM, DI = diabetic IFM; n = 4. All mitomiRs reported in the heat map are expressed as normalized intensities. (B) QRT-PCR analyses of miR-378 in control and diabetic whole heart tissue. Values are represented as mean ± SE, n = 4. U6 mRNA served as control. (C) QRT-PCR analyses of mitomiR-378 in SSM and IFM diabetic mitochondrial subpopulations as compared with control. Values are represented as mean ± SE, n = 6. U6 mRNA served as control. (D) QRT-PCR analyses for pre-miR-378 in SSM and IFM diabetic mitochondrial subpopulations as compared with control. Values are represented as mean ± SE, n = 6 for SSM and IFM. AMA mRNA served as a reference control; cytoplasm served as a positive control; no template served as a negative control.

Figure 3

MitomiR-378 and its targeting to the mitochondrial genome. (A) Schematic representation of miR-378 location in the PPARGC1b (PGC-1b) gene. (B) QRT-PCR quantification of PGC1α and PGC1β transcripts in control and diabetic hearts. GAPDH served as control. Values are represented as mean ± SE; n = 4 for each group. *P < 0.05 for Control vs. Diabetic. (C) Evaluation of the minimum free energy (MFE) value that characterizes the stability of mitomiR-378/mRNA interaction, identified by MicroCosm and RNAhybrid. (D) Relative luciferase activities of ATP6 reporter co-expressed with miR-378, miR-200c, or plasmid (pCMV-MIR) control in HEK293 cells 24 hours post transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. Values are represented as mean ± SEM; n = 7 per group. *p<0.05 for miR-378 vs. all other groups. (E) Western blot analysis of ATP6 in control and diabetic SSM and IFM. Cox IV serves as a loading control. Values are represented as mean ± SE; n = 6. *p<0.05 for Control vs. Diabetic. (F) ATP synthase activity expressed in activity/min/mg protein. Values are means ± SE; n = 8 for each group. *P < 0.05 for Control IFM vs. Diabetic IFM.

Figure 4

Redistribution of RISC components in the mitochondrion. (A) Western blot analyses of Ago2 and FXR1 in control and diabetic whole heart. GAPDH serves as loading control. (B) Quantification of Ago2 protein content in control and diabetic whole heart. Values are means ± SE; n = 3. (C) Quantification of FXR1 protein content in control and diabetic whole heart. Values are means ± SE; n = 4. GAPDH served as loading control. Western blot analyses of Ago2 and FXR1 in Control and Diabetic (D) SSM and (E) Control and Diabetic IFM. Quantification of (F) Ago2 and (G) FXR1 protein content in control and diabetic mitochondrial subpopulations. Values are means ± SE; n = 3. COX IV serves as a loading control.

Figure 5

Crosslinked immunoprecipitation (CLIP) in cardiac mitochondrial subpopulations and MitomiR-378 RISC constituent interactions with the mitochondrial genome. (A) Western blots of biotinylated RNA from CLIP-Ago2 and CLIP-FXR1 reactions illustrating crosslinked protein/RNA and the associated gel shift from 80 kDa to 95–110 kDa. (B) Western blot analyses of CLIP-Ago2 and CLIP-FXR1 subjected to RNAase I treatment at 1:50 dilution (high RNAase) illustrating interaction between the two proteins in the absence of RNA. (C) CLIP-Ago2 and CLIP-FXR1 associated enrichment analyses of mitomiR-378 analyzed by qRT-PCR in control and diabetic cardiac mitochondrial subpopulations. Values are presented as means ± SE; n = 2 where each individual sample represents a pool of 5 individual animals. (D) CLIP-Ago2 and CLIP-FXR1 associated enrichment analysis of transcripts for mitochondrial encoded ATP6 mRNA levels as assessed by qRT-PCR analysis in control and diabetic cardiac mitochondrial subpopulations. Values are presented as means ± SE; n = 2 where each individual sample represents a pool of 5 individual animals.

Figure 6

Genomic sequencing analyses of mitochondrial RISCome association with mitochondrial mRNA and mitomiRs. (A) Mitochondrially-encoded mRNAs in the diabetic subpopulations which were differentially expressed in the mitoRISCome relative to controls. (B) Mapping of the footprint regions of mitochondrial mRNA sequence identified by next generation sequencing which were present in the mitoRISCome. (C) Genome browser illustration of mitochondrial RNA-seq reads mapping to rRNA, tRNA, mRNAs; histogram indicates RNA-seq read distribution which includes the transcript region. (D) MitomiR heat map derived from next generation sequencing of small RNAs identifying enrichment and depletion patterns within the mitoRISCome of diabetic SSM and diabetic IFM, relative to respective controls. (E, F) Mapping of the location of the footprint regions of mitomiR sequence identified by next generation sequencing which were present in the mitoRISCome and were enriched with tri-nucleotide motifs, AGG (red blocks) or UGG (yellow blocks). (G) Mapping of mitoRISCome associated mitomiR-378 enrichment clusters sites in control and diabetic SSM and IFM. (H) MitoRISCome:mitomiR378 ternary maps for the ATP6 target in the diabetic IFM.

Figure 6

Genomic sequencing analyses of mitochondrial RISCome association with mitochondrial mRNA and mitomiRs. (A) Mitochondrially-encoded mRNAs in the diabetic subpopulations which were differentially expressed in the mitoRISCome relative to controls. (B) Mapping of the footprint regions of mitochondrial mRNA sequence identified by next generation sequencing which were present in the mitoRISCome. (C) Genome browser illustration of mitochondrial RNA-seq reads mapping to rRNA, tRNA, mRNAs; histogram indicates RNA-seq read distribution which includes the transcript region. (D) MitomiR heat map derived from next generation sequencing of small RNAs identifying enrichment and depletion patterns within the mitoRISCome of diabetic SSM and diabetic IFM, relative to respective controls. (E, F) Mapping of the location of the footprint regions of mitomiR sequence identified by next generation sequencing which were present in the mitoRISCome and were enriched with tri-nucleotide motifs, AGG (red blocks) or UGG (yellow blocks). (G) Mapping of mitoRISCome associated mitomiR-378 enrichment clusters sites in control and diabetic SSM and IFM. (H) MitoRISCome:mitomiR378 ternary maps for the ATP6 target in the diabetic IFM.

Figure 7

Validation of miR-378 mitochondrial targeting in vitro. (A) RT-PCR analyses for miR-378 levels in isolated mitochondria from HL-1 (Control) and miR-378 cells; n = 3. (B) QRT-PCR analyses for ATP6 mRNA in isolated mitochondria from Control and miR-378 cells; n = 4. (C) Representative Western blot analyses of ATP6 and GW182 protein levels in isolated mitochondria from Control and miR-378 cells. COX IV protein expression is utilized as a loading control. (D) Quantitative analysis of ATP6 protein levels in isolated mitochondria from Control and miR-378 cells. Values are expressed per COX IV protein levels; n = 6. (e) ATP synthase activity in control HL-1 cells and in miR-378 cells; n = 4. Values are means ± SE. *P < 0.05 for Control vs. miR-378.

Figure 8

Validation of miR-378 mitochondrial targeting in vivo. Quantitative analyses by Western blot of ATP6 protein levels following LNA-miR-378 treatment and diabetic induction in (A) SSM and (B) IFM. COX IV protein expression is utilized as a loading control and values are expressed per COX IV. ATP synthase activities levels following LNA-miR-378 treatment and diabetic induction in (C) SSM and (D) IFM. For ATP6 and ATP synthase analyses. Values are means ± SE, n = 5. *P < 0.05 for Control vs. Diabetic and Control vs. Scrambled. (E) Representative M-mode images of Control, Diabetic, Scrambled and LNA-miR-378 treated hearts 5 weeks following diabetes mellitus induction. (F) Quantitative summary of % ejection fraction and (G) % fractional shortening prior to (baseline) and 5 weeks following diabetes mellitus induction. Values are means ± SE, n = 5. *P < 0.05 for baseline vs. 5 weeks; #P < 0.05 for LNA-miR-378 vs. Scrambled. ScR = Scrambled; miR = LNA-miR-378.