MicroRNA-425 and microRNA-155 cooperatively regulate atrial natriuretic peptide expression and cGMP production

Abstract

Aims: Atrial natriuretic peptide (ANP), secreted primarily by atrial cardiomyocytes, decreases blood pressure by raising cyclic 3',5'-guanosine monophosphate (cGMP) levels and inducing vasorelaxation, natriuresis, and diuresis. Raising the level of ANP has been shown to be an effective treatment for hypertension. To advance the future development of an anti-microRNA (miR) approach to increasing expression of ANP, we investigated the regulation of NPPA expression by two miRs: miR-425 and miR-155. We examined whether miR-425 and miR-155 have an additive effect on the expression and function of ANP.

Methods and results: Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) were transfected with miR-425, miR-155, or a combination of the two miRs. Two days later, NPPA expression was measured using real time qPCR. Each of the miRs decreased NPPA expression over a wide range of concentrations, with a significant reduction at concentrations as low as 1 nM. The combination of miR-425 and miR-155 reduced NPPA expression to a greater extent than either miR-425 or miR-155 alone. An in vitro assay was developed to study the potential biological significance of the miR-induced decrease in NPPA expression. The cooperative effect of miR-425 and miR-155 on NPPA expression was associated with a significant decrease in cGMP levels.

Conclusions: These data demonstrate that miR-425 and miR-155 regulate NPPA expression in a cooperative manner. Targeting both miRNAs with anti-miRs (possibly at submaximal concentrations) might prove to be a more effective strategy to modulate ANP levels, and thus blood pressure, than targeting either miRNA alone.

Fig 1. MiR-425 and miR-155 produced dose-dependent decreases in NPPA expression over a wide range of concentrations.

NPPA mRNA levels in human embryonic cell cardiomyocytes (hESC-CMs) transfected with 1 nM, 5 nM, 10 nM, or 20 nM of negative control miRNA (NC), miR-425, or miR-155. Expression is normalized to GAPDH expression and shown relative to expression in cells transfected with negative control miRNA. *P<0.01 and **P<1x10^-6 versus cells transfected with the same concentration of negative control miRNA. N = 4–8 experiments per miRNA concentration (4–12 replicate wells per condition for each experiment).

Fig 2. MiR-425 and miR-155 had an additive repressive effect on cardiomyocyte NPPA expression.

NPPA mRNA levels in hESC-CMs that were transfected with either miR-425 or miR-155 alone or with a combination of miR-425 and miR-155. Negative control miRNA (NC) was used as needed to make the total concentration of miRNA constant in each condition. NPPA expression is shown relative to that in cells transfected with negative control miRNA. *P<0.01 and **P<1x10^-6 versus cells transfected with negative control miRNA. N = 3 experiments (6–12 replicate wells per condition).

Fig 3. The combination of miR-425 and miR-155 resulted in greater repression of NPPA expression than a higher concentration of either miRNA alone.

Cardiomyocytes were transfected with 1 nM (A) or 5 nM (B) of miR-425, miR-155, or both miR-425 and miR-155, each at half the concentration of either miRNA alone. Two days later, NPPA mRNA levels were measured. NPPA expression was normalized to GAPDH expression, and shown relative to expression in cells transfected with the negative control miRNA. *P<0.01 and **P<1x10^-6 versus cells transfected with negative control miRNA. N = 4 experiments (4–12 replicate wells per condition) for (A) and n = 8 experiments (4–12 replicate wells per condition) for (B).

Fig 4. Development and validation of a biological assay to measure the effects of miRNA-mediated changes in NPPA expression on cGMP levels.

TurboGFP (tGFP) fluorescence in COS7 cells 48 hours after transfection with the NPR1-tGFP expression vector (A). Cell nuclei were visualized using DAPI. The effect of incubating NPR1-expressing COS7 cells with increasing amounts of human ANP on cGMP concentrations (B). Cyclic GMP levels were expressed as picomoles of cGMP per mg of protein, relative to the cGMP levels at baseline (cells not exposed to ANP). The correlation between cGMP levels in NPR1-expressing COS7 cells exposed to media from cardiomyocytes transfected with miRNA(s), and cardiomyocyte NPPA mRNA levels (C) or Nt-proANP levels in the cardiomyocyte media (D).

Fig 5. MiR-425 and miR-155 produced an additive decrease in cGMP levels relative to either miRNA alone.

Cardiomyocytes were transfected with 1 nM (A) or 5 nM (B) of negative control miRNA (NC), miR-425, miR-155, or the combination of miR-425 and miR-155, each at half the concentration of the other miRs. Cyclic GMP levels were measured in NPR1-expressing cells that were incubated for 2 hours with media collected from the cardiomyocytes. Cyclic GMP levels were expressed as picomoles of cGMP per mg of protein and were shown relative to levels in cells exposed to media from cardiomyocytes transfected with negative control miRNA. *P<0.01 and **P<1x10^-6 versus cells transfected with negative control miRNA. N = 3 experiments (4–12 replicate wells for each condition) for (A) and n = 8 experiments (4 replicate wells per condition) for (B).