Silencing the lncRNA Chast Reverses Cardiac Disease

Prof. Thomas Thum and Dr. Janika Viereck work at the Institute of Molecular and Translational Therapeutic Strategies, Hannover Medical School, Germany. They recently published a Science Translational Medicine paper on the lncRNA Chast and its involvement in cardiac remodelling. They have been using Antisense LNA GapmeRs in vivo to silence Chast and reverse cardiac disease.

You recently published a great paper in Science Translational Medicine on the involvement of lncRNAs in cardiac remodeling. Can you briefly summarize your findings?

We were interested in identifying long non-coding RNAs (lncRNAs) that provide novel promising treatment options for cardiovascular disease. By lncRNA profiling, we identified a transcript that was strongly induced in hearts, and especially in cardiomyocytes of an experimental mouse model for pressure overload-induced cardiac hypertrophy and heart failure.

We named this lncRNA Cardiac hypertrophy associated transcript (Chast). Chast acts downstream of the pro-hypertrophic NFAT signaling pathway and drives pathological changes in the heart by blocking autophagy, which is an important recycling process for the recovery of nutrients and cellular components.

Pharmacological inhibition of Chast by Antisense LNA GapmeRs both prevented and attenuated the abnormal growth of the murine myocardium and fibrotic remodeling and significantly improved the heart’s pump function, with no significant changes in toxicological parameters.

Inhibition of lncRNA Chast by Antisense LNA GapmeRs significantly improved the heart’s pump function [in our mouse model].

In accordance with the mouse model of cardiac hypertrophy, we identified a human homolog that was significantly induced in hypertrophic hearts from aortic stenosis patients and in human embryonic stem cells-derived cardiomyocytes upon hypertrophic stimulation. Furthermore, over-expression of the human Chast in murine cardiomyocytes induced the growth of these cells, indicating a functional conservation of Chast activity.

This sets the ground for future pre-clinical and clinical development of Chast inhibitors for the treatment of pathological cardiac remodeling, such as cardiac hypertrophy and potentially heart failure.

What made you focus on the Chast lncRNA?

First of all, we aimed to focus specifically on up-regulated lncRNAs, because we were interested in developing inhibitory drugs as a therapeutic approach. Interestingly, Chast expression was elevated not only at the time point investigated, but also remained elevated over the time of hypertrophic remodeling. Furthermore, we observed this induction specifically in cardiomyocytes, suggesting an important role in cardiac hypertrophy.

Could you describe how you discovered that the lncRNA Chast induces cardiomyocyte hypertrophy?

We performed experiments in vitro and in vivo, in which we manipulated Chast expression, and assessed the effects on cardiomyocyte growth (the hallmark of cardiac hypertrophy), and expression of hypertrophic marker genes.

In cultured cardiomyocytes, overexpression based on lentiviral transduction led to an enlargement of cardiomyocyte size, and increased expression of the hypertrophic marker Anp. Silencing of Chast with Antisense LNA GapmeRs in cardiomyocytes treated with prohypertrophic factors had the opposite effect of attenuating cell growth and Anp expression.

For in vivo studies, we injected mice with AAV9 particles harboring the Chast sequence under the control of a cardiomyocyte-specific promoter. This led to hypertrophic growth of the heart with a significant gain of heart weight and size, increase in cardiomyocyte size, and enhanced expression of hypertrophic markers.

In contrast, treatment with Chast Antisense LNA GapmeRs effectively prevented and was even capable of reversing experimentally induced hypertrophy in mouse models of cardiac disease.

Altogether, these results demonstrate that Chast plays an important role in promoting hypertrophy of the heart.

Treatment with Chast Antisense LNA GapmeRs prevented and even reversed experimentally induced hypertrophy.

What you have learnt about the function of the lncRNA Chast so far?

Chast is encoded antisense to Plekhm1, a recently described regulator of autophagy. Since the expression of both genes was inversely correlated over the time course of cardiac remodeling, and Plelkhm1 repression affected cardiac hypertrophy, we assumed that Chast regulates Plekhm1 expression by a so far unknown antisense mechanism.

Chast is detectable in the nuclear as well as the cytoplasmic compartment of cardiomyocytes, so it might have additional biological functions. This is supported by global mRNA expression changes upon Chast repression, and protein interaction studies, unravelling an impact of this lncRNA on pathological cardiac pathways as well as proteins involved in cardiomyopathies.

Is the lncRNA Chast relevant to human biology and cardiac disease?

lncRNAs are in general poorly conserved among species. Intriguingly, we could identify a human sequence that was similar in sequence and structure.

In accordance with the murine transcript, human Chast was up-regulated in embryonic stem cell-derived cardiomyocytes upon hypertrophic stimuli as we as in heart tissue from aortic stenosis patients, highlighting a translational relevance of Chast lncRNA.

You used Antisense LNA GapmeRs to achieve knockdown of Chast in cell cultures and to prevent cardiomyocyte overexpression of Chast in mice in response to cardiac pressure overload. In your opinion, what are the advantages of Antisense LNA GapmeRs over RNAi-based methods and Crispr-Cas9 technology when it comes to loss of function analysis of lncRNAs?

Antisense LNA GapmeRs have the advantage of tackling lncRNA activities directly at the location of their biosynthesis – in the nucleus. This is especially of interest for studies on transcripts that remain and function in the nucleus. RNAi-based methods might not sufficiently repress such lncRNAs.

Knockout strategies by genome editing for lncRNAs like Chast (that are located antisense to protein-coding genes), or for enhancer-derived transcripts, might generate results that remain open to interpretation. If an effect is observed, it might be impossible to tell whether it is mediated by knockout of the lncRNA itself, or by parts of the protein-coding gene or enhancer region.

Antisense LNA GapmeRs target the lncRNA directly and allowed us to test the therapeutic potential of Chast silencing in animal models.

Antisense LNA GapmeRs directly target the lncRNA. In addition, Antisense LNA GapmeRs can be taken up by cells in an unassisted manner (gymnosis). That is of advantage for cells that are difficult to transfect or that are sensitive to certain transfection reagents.

Crucial to our work, these Antisense LNA GapmeRs also allowed us to test the therapeutic potential of Chast silencing directly in animal models of cardiac disease.

Can you briefly tell us about your future plans for work on the Chast lncRNA?

We are currently in more depth evaluating the Chast function in cardiomyocytes and testing human Chast Antisense LNA GapmeRs in human cardiomyocytes with the aim to set a pathway for future development of clinical strategies applying Chast inhibitors in humans with pathological remodeling.

This sets the ground for development of Chast inhibitors for the treatment of cardiac hypertrophy and potentially heart failure.

Viereck et al. Long noncoding RNA Chast promotes cardiac remodeling. Sci Transl Med. 2016 Feb 17;8(326):326ra22. PMID: 26888430.