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Final Report Summary - SEARCH (a Study of the Epigenetic Alterations that Result in Cardiac Hypertrophy)

Cardiovascular diseases are the leading cause for mortality and morbidity in the developed world, but despite their manifest importance, the underlying causes are still poorly understood. We have studied one of the features associated with many of these diseases (e.g. high blood pressure, aortic valve stenosis, cardiac infarction), cardiac hypertrophy [CH]. CH is a response of the heart to an increase in demand by an increase in size of the constituent cardiomyocytes. A similar response is observed under certain physiological conditions (e.g. upon chronic exercise, normal adolescent growth or pregnancy). Interestingly however, physiological CH is beneficial and readily reversible, whereas pathological CH is detrimental and irreversible. This irreversibility presents a major challenge in the management of patient with pathological CH, and is mainly considered to be a consequence of cardiac fibrosis. Although pathological hypertrophy is indeed associated with an increase in the number of cardiac fibroblasts and a decrease in cardiomyocytes, preliminary data from the host laboratory suggested also a cardiomyocytes-specific, cell-autonomous irreversibility. We hypothesised that epigenetic changes upon CH were at the basis of the altered phenotypic flexibility of cardiomyocytes.
Because cardiomyocytes are the cell-type responsible for CH but make up only ~25% of the heart’s cells, we chose to introduce a technique to look at cardiomyocytes only. We successfully managed to isolate nuclei from the heart and separate myocyte from non-myocyte nuclei using a flow-sorter[Bergmann]. Using this method, we studied nuclei from rat hearts that had been subjected to chronic exercise (physiological CH) or pressure overload by aortic banding (pathological hypertrophy), together with their respective controls (n=4 for each). Both treatments resulted in a similar degree of hypertrophy (~50% increase in heart-body weight ratio). Nuclei flow sorting analysis demonstrated a 25% reduction in the number of myocytes in response to aortic banding and - interestingly - a 16% increase in response to chronic exercise. The latter suggests a moderate hyperplasia in addiction to hypertrophy, and is in correspondence with a recent report suggesting induction of cardiomyocytes proliferation upon exercise [Bersel]. Pure myocyte nuclei (>95%) were used as a source for RNA, DNA and chromatin.
To obtain genome-wide transcriptome and epigenome profiles, we employed high-throughput sequencing: nuclear RNA was converted into sequencing libraries following the dUTP-method, cytosine methylation in the DNA was profiled using meDIP-seq, and dimethylation of lysine 9 and trimetylation of lysine 27 on histone 3 (resp. H3K9me2 and H3K27me3) were profiled by native chromatin immunoprecipitation followed by library construction and sequencing (nChIP-seq). DNA and chromatin input controls were also sequenced, totalling 112 libraries. A preliminary ontology analysis of the transcriptome data showed significant enrichment of cardiomyocytes-specific processes like cardiac muscle contraction (40-fold; p=1.6E-10) and contractile fibers (26-fold; p= 2.85E-12), demonstrating fidelity of the approach. A full data analysis of these profiles is underway and results will be the subject of further studies in the host laboratory. A number of interesting observations were already made. These include a plethora of novel non-coding RNAs, some of which alter in expression with cardiac phenotype; the inverse correlation between gene body methylation and gene expression which contrasts with the situation observed in most cell types; and the first evidence for targeted mRNA transport in cardiomyocytes.
In a parallel set of experiments we employed a set of small molecules to inhibit specific epigenetic enzymes responsible for methylation of cytosine on DNA or of lysines 9 or 27 on histone 3. These are deazaneplanocine (DZnep, inhibits the Enhancers of Zeste 1 and 2), RG108 (inhibits DNA methyltransferases 1, 3a and 3b), BIX01294 and UNC0638 (inhibit G9a and G9a-like protein - GLP), Chaetocin (inhibits Suppressors of Variegation 39h1 and 39h2). We added these to primary cultures of neonatal rat ventricular myocytes (NRVMs) treated with endothelin-1 (ET-1) or insulin-like growth factor 1 (IGF1) to induce respectively pathological or physiological hypertrophy in vitro. Hypertrophy was assessed by tritiated leucine incorporation, ANF protein expression and the expression a panel of hypertrophic marker genes (Myh6, Myh7, Nppa, Nppb, Acta1 and Tnnt2) for physiological and pathological hypertrophy. Some of these inhibitors on their own were sufficient to induce an increase in protein synthesis and increased ANF protein expression. Marker gene profiling demonstrated an increased expression of Tnnt2 and Acta1, which are markers of general hypertrophy. We moreover showed an increase in Myh7 but not Myh6 expression as well as of Nppa and Nppb expression, demonstrating a reactivation of the fetal gene program, a hallmark of pathological hypertrophy. We thus concluded that inhibition of some epigenetic repressors induce spontaneous pathological hypertrophy in NRVM.

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United Kingdom
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