Micro-RNA-195 and -451 regulate the LKB1/AMPK signaling axis by targeting MO25

Abstract

Background: Recently, MicroRNAs (miR) and AMP-kinase (AMPK) have emerged as prominent players in the development of cardiac hypertrophy and heart failure. We hypothesized that components of the adenosine monophosphate-activated kinase (AMPK) pathway are targeted by miRs and alter AMPK signaling during pathological cardiac stress.

Methodology/principal findings: Using a mouse model of hypertrophic cardiomyopathy (HCM), we demonstrated early elevation of miR-195 and miR-451 in HCM hearts, which targets MO25, a central component of the MO25/STRAD/LKB1 complex that acts as an upstream kinase for AMPK. We show functional targeting of MO25 by miR-195 and -451. Further in vitro interrogation of MO25 as a functional target validated this hypothesis where over-expression of miR-195 in C2C12 cells knocked down MO25 expression levels and downstream AMPK signaling (phosphorylation of Acetyl CoA carboxylase [ACC] and AMPK activity assay), similar to MO25 knockdown in C2C12 cells by siRNA. Parallel changes were measured in 60 day R403Q HCM male hearts that were rescued by short-term administration of AICAR, an AMPK agonist.

Conclusions/significance: Elevated miR-195 targets the LKB1/AMPK signaling axis in HCM progression and implicates a functional role in HCM disease progression. MiR-195 may serve as potential therapeutics or therapeutic targets for heart disease.

Figure 1. miR-195 and miR-451 expression in WT and R403Q HCM hearts.

A: Expression levels determined by RT-PCR of miR-195; B: miR-451 (right panel) in 60, 120, and 240 day male WT and R403Q HCM hearts corrected by U6 expression (n = 3 for each group, *p<0.05), C: Northern blot analysis of miR-195 and miR-451 expression in WT and R403Q HCM hearts. Northern blot analysis using LNA-modified, 5′ end biotin-conjugated prob1es for U6 RNA, miR-195 and miR-451 using RNA extracted from 60-day- and 120-day-old WT and R403Q HCM hearts. Each lane represents one sample of extracted RNA pooled together from 4 total hearts. Northern blot images in C are taken from the same blot but were cropped for clarity.

Figure 2. In situ hybridization of miR-195 and miR-451 expression in R403Q HCM hearts.

In situ hybridization of miR-195 and miR-451 in the left ventricle of male R403Q HCM hearts. 5′ digoxin-conjugated LNA-modified DNA probes complementary to mature miR-195 or -451 were used to detect native miR-195 and -451. Panel A (100X magnification): Scrambled sequence, missense 5′ digoxin-conjugated LNA-modified DNA probe; Panel B (100X magnification): miR-195, 5′ digoxin-conjugated LNA-modified miR-195 sequence probe; Panel C (100X magnification): miR-451, 5′ digoxin-conjugated LNA-modified miR-451 sequence probe. Insets: 200 X magnification. No significant signal was detected in hearts probed with the scrambled sequence. Sections treated with probes specific for miR-195 or miR-451 demonstrated a detectable signal.

Figure 3. MiRs, including miR-195 and -451, target prediction (Targetscan) in murine MO25 3′ UTR.

Top panel: Putative binding sites for miR-195 and miR-451 (underlined) are highlighted in red. Bottom panel: Sequence alignment (indicated in boldface and large lettering) of putative miR-195 and miR-451 binding sites in 3′ UTR of MO25 of several species, showing a high level of sequence conservation.

Figure 4. MO25 is a functional target of miR-195 and miR-451.

A: C2C12 cells transfected with luciferase constructs. MO25 3′ UTR target sequences (as indicated) of miR-195 (left panel) or miR-451 (right panel). Bar graph representation of luciferase expression in C2C12 cells treated with miR-195 (left panel) or miR-451 (right panel) mimics or negative siRNA control. Each bar indicates mean values ± S.E.M. of 3 separate experiments. *P<0.05 compared to WT group. B: Increase in expression of MO25 in C2C12 cell by miScript miRNA inhibitor delivered using HiPerfect reagent. AllStars Negative Control siRNA was used as negative control. Each bar indicates mean values ± S.E.M. of 3 separate experiments. *P<0.05 compared to WT group.

Figure 5. MO25 expression and downstream AMPK signaling in C2C12 cells.

A, Left panel: Bar graph summary (top panel) of Western blots (bottom panel) for MO25, acetyl CoA carboxylase (ACC) and phosphorylated ACC (p-ACC) in C2C12 cells transfected with either 0 ng, 200ng or 400ng of miR-195 overexpression constructs. Each bar indicates mean values ± S.E.M. of 3 separate experiments and represents the ratio of total protein to GAPDH or phosphorylated protein to each respective level of total protein following normalization to GAPDH. *P<0.05 compared to 0 ng C2C12 cell group. A, Right panel: Bar graph summary (top panel) of Western blots (bottom panel) for MO25, acetyl CoA carboxylase (ACC) and phosphorylated ACC (p-ACC) in C2C12 cells treated with Stealth RNAi siRNA for miR-195 using HiPerfect reagent. AllStars Negative Control siRNA was used as negative control. Each bar indicates mean values ± S.E.M. of 3 separate experiments and represents the ratio of total protein to GAPDH or phosphorylated protein to each respective level of total protein following normalization to GAPDH. *P<0.05 compared to control group. B: miR-195 overexpression and MO25 siRNA knock-down significantly decreased AMPK activities in direct AMPK Kinase assay (Cyclex), While AICAR increased AMPK activities. Each plot indicates mean values ± S.E.M. of 3 separate experiments. *P<0.05 compared to control group; § P<0.05 compared to miR-195 or siRNA knock-down group.

Figure 6. Modification of AMPK signaling axis in R403Q HCM hearts.

Bar graph summary (top panel) of Western blots (bottom panel) for MO25, AMPK, phosphorylated AMPK (p-AMPK), ACC and p-ACC using left ventricular lysates from 60 day male WT or R403Q HCM hearts. Each bar indicates mean values ± S.E.M. (n = 6 hearts) and represents ratio of total protein to GAPDH or phosphorylated protein to each respective level of total protein following normalization to GAPDH. *P<0.05 compared to WT controls.

Figure 7. Modification of AMPK signaling axis in R403Q HCM hearts following five-day administration of AICAR.

A: Bar graph summary (top panel) of Western blots (bottom panel) for MO25, AMPK, phosphorylated AMPK (p-AMPK), ACC and p-ACC using left ventricular lysates from 60 day male WT or R403Q HCM hearts with or without AICAR administration. Each bar indicates mean values ± S.E.M. (n = 3 hearts) and represents ratio of total protein to GAPDH or phosphorylated protein to each respective level of total protein following normalization to GAPDH. *P<0.05 compared to controls. B: AMPK activities in WT or R403Q HCM hearts following five-day administration of AICAR. Each plot indicates mean values ± S.E.M. of 5 separate experiments. *P<0.05 compared to respective control group; § P<0.05 compared to WT control group. C: Relative expression of miR-195 and miR-451 in WT or R403Q HCM hearts following AICAR treatment. Expression levels were determined by RT-PCR and illustrated relative to WT saline injected animals. Data are presented as the mean values ± S.E.M. of 4–5 samples. *P<0.05 compared to respective control group; § P<0.05 compared to WT control group.

Figure 8. Predicted model of miR-195/mi-R451 regulation of the AMPK signaling axis in R403Q HCM hearts.

The functional consequence of the R403Q mutation in the heart is an increase in the relative amount of AMP and ADP. This, in turn, will induce a hypertrophic stress that will elevate expression of miR-195 and miR-451. In addition, the energetic stress caused by this mutation will drive an elevation in AMPK signaling. Based on our findings, the net result is that hypertrophic stress overrides the energetic stress (large arrow) such that miR-195-dependent reduction in MO25 expression leads to a decrease in AMPK signaling indicated by decreased levels of p-ACC.