Antisense regulation of atrial natriuretic peptide expression
Selvi Celik, Mardjaneh Karbalaei Sadegh, Michael Morley, Carolina Roselli, Patrick T. Ellinor, Thomas Cappola, J. Gustav Smith, Olof Gidlöf
Selvi Celik, Mardjaneh Karbalaei Sadegh, Michael Morley, Carolina Roselli, Patrick T. Ellinor, Thomas Cappola, J. Gustav Smith, Olof Gidlöf
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Research Article Cardiology

Antisense regulation of atrial natriuretic peptide expression

Abstract

The cardiac hormone atrial natriuretic peptide (ANP) is a central regulator of blood volume and a therapeutic target in hypertension and heart failure. Enhanced ANP activity in such conditions through inhibition of the degradative enzyme neprilysin has shown clinical efficacy but is complicated by consequences of simultaneous accumulation of a heterogeneous array of other hormones. Targets for specific ANP enhancement have not been available. Here, we describe a cis-acting antisense transcript (NPPA-AS1), which negatively regulates ANP expression in human cardiomyocytes. We show that NPPA-AS1 regulates ANP expression via facilitating NPPA repressor RE1-silencing transcription factor (REST) binding to its promoter, rather than forming an RNA duplex with ANP mRNA. Expression of ANP mRNA and NPPA-AS1 was increased and correlated in isolated strained human cardiomyocytes and in hearts from patients with advanced heart failure. Further, inhibition of NPPA-AS1 in vitro and in vivo resulted in increased myocardial expression of ANP, increased circulating ANP, increased renal cGMP, and lower blood pressure. The effects of NPPA-AS1 inhibition on NPPA expression in human cardiomyocytes were further marked under cell-strain conditions. Collectively, these results implicate the antisense transcript NPPA-AS1 as part of a physiologic self-regulatory ANP circuit and a viable target for specific ANP augmentation.

Authors

Selvi Celik, Mardjaneh Karbalaei Sadegh, Michael Morley, Carolina Roselli, Patrick T. Ellinor, Thomas Cappola, J. Gustav Smith, Olof Gidlöf

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Figure 7

Mechanical strain increases NPPA and NPPA-AS1 expression in cardiomyocytes.

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Mechanical strain increases NPPA and NPPA-AS1 expression in cardiomyocyt...
(A) Overview of the setup and design of the strain experiment. (B) NPPA and NPPA-AS1 expression during the time course of the experiment quantified by qRT-PCR. Expression is presented relative to GAPDH and normalized to the mean of the cells at time point 0 hours. Results are based on 3 separate experiments with 3 replicates in each group. Mean and standard deviation are shown. The Kruskal-Wallis test was used to test the difference in expression between baseline and each time point. *P < 0.05, ***P < 0.001 after adjustment for multiple comparisons using Dunn’s test. (C) Protein levels of ANP at baseline and after 48 hours of stretch. Results are expressed relative to GAPDH protein levels. Data are from 2 separate experiments with 3–4 replicates in each group. Shown are mean and standard deviation in each group. *P < 0.05 by Mann-Whitney U test. Shown below are representative blots for NPPA and GAPDH. US, unstretched; S, stretched. See complete unedited blots in the supplemental material. (D) NPPA expression in iPS-CMs first transfected with siRNA against NPPA-AS1 and then subjected to 48 hours of stretch. Two-way ANOVA with Tukey’s multiple-comparisons test was used to test differences within and between groups.*P < 0.05, **P < 0.01, ***P < 0.001. (E) RNA-Seq expression data for NPPA and NPPA-AS1 in ventricular tissue from heart failure patients (n = 42) and unused donor hearts (n = 22). ***P < 0.001 by Mann Whitney U test. (F) Correlation of NPPA and NPPA-AS1 in left ventricle and left atrium from heart failure and nonfailure donors. Pearson’s correlation coefficient and P value are shown.