| dc.description.abstracteng | Cardiovascular diseases (CVDs) are the leading cause of hospital admission and mortality
worldwide. Heart failure (HF) is one of the most common CVDs and is characterized by a
reduced cardiac function and left ventricular dilatation resulting in the inability of the heart to
supply sufficient blood-flow. Upon cardiac stress, for example by heart valve defects or
pressure overload (PO) from aortic stenosis, growth of cardiomyocytes (CMs) occurs. This
initial hypertrophic (HT) adaptation preserves cardiac performance. Persistent hemodynamic
stress leads to decompensation of the heart resulting in HF. Progression to HF is accompanied
by underlying molecular, cellular, and interstitial changes, termed cardiac remodeling.
Treatment options concentrate on inhibition of neuroendocrine stimulation or deregulated
signaling pathways, but this has only limited efficacy and the morbidity and mortality of HF
patients remains high. Therefore, other therapeutic options need to be investigated. Aberrant
gene expression, such as re-expression of the fetal gene program, is a key-event in HF
progression. Recent research suggested that changes in gene expression are not only
regulated by transcription factors, but also by epigenetic processes such as non-coding RNAs
and histone or DNA modifications. For example, DNA methylation and histone modifications
such as acetylation or methylation can influence transcription of cardio-specific gene
programs. Another less studied epigenetic modification is RNA methylation. Similar to DNA,
RNA can be post-transcriptionally modified. The most prevalent modification is methylation of
the adenosine base, termed m6A methylation. This m6A methylation is a dynamic and
reversible process, mediated by so-called ‘writer’ and ‘eraser’ proteins adding or removing
methylation marks on RNA. m6A methylation has been found in all classes of RNA, including
mRNA, rRNA, tRNA, snRNA, and miRNA. Recent research has suggested that RNA
methylation can affect splicing, mRNA transport, translation, storage, or decay. These effects
were shown to be either mediated directly by conformational changes of methylated RNA or
by so called ‘reader’ proteins, which recognize methylation marks. This adds an additional
layer of transcriptional and translational control.
m6A RNA methylation was first discovered in the 1970s, but long thought to be of minor
importance. Newly developed methods to detect and map m6A methylation in 2012 allowed
for more thorough investigation of the modification and gave rise to the term ‘epitranscriptome’,
describing the RNA methylation patterns which govern RNA regulation. The epitranscriptome
has since been studied intensively in various diseases and fields of biology, including many
forms of cancer, neurodegenerative diseases, and developmental biology. However, little is
known about the role of m6A methylation in cardiac diseases such as HT and HF. Only recently
have a few publications described RNA methylation in ischemic cardiac tissue and hypoxia, or the deregulation of m6A methylation in response to hypertrophic stimuli in cell culture. Until
now, the global epitranscriptome of the heart has never been described.
In my doctoral work, I have analyzed and characterized the global epitranscriptome of healthy
human and mouse heart tissue. My analysis demonstrated that a considerable number of
detected transcripts carried m6A methylation marks. Furthermore, many of the identified
methylated transcripts were found in both mouse and human tissue and were of cardiac
specificity, underlining the importance of this RNA modification in cardiac tissues. To better
understand the role of these modification in cardiac HT and HF, I analyzed m6A RNA
methylation changes in a mouse model of PO as well as in cardiac tissue from human endstage
HF patients. Interestingly, many more transcripts were changed at the methylation level
than on the expression level. Furthermore, transcripts altered at the methylation level tend to
code for proteins participating in metabolic, catabolic and signal transduction pathways,
whereas transcripts with altered expression generally code for completely different pathways,
such as those involved in cardiac plasticity and remodeling. Since many transcripts were only
altered at the methylation level, polysomal profiling was applied to elucidate if m6A methylation
impacts translation, which indeed was the case. From this analysis, I propose a new
mechanism of transcription independent translation regulation by RNA methylation. I could
validate that protein levels change in correlation with altered methylation while the expression
level remains unchanged. Furthermore, I hypothesize a mechanism of fast-translation with
fast-turnover mediated by m6A methylation which allows CMs to adapt to stressful conditions.
Further, I investigated the effect of manipulation of the RNA demethylase FTO on hypertrophic
responses in vitro and in -vivo. For in vitro studies, a human induced pluripotent stem cell
(iPSC) model was used. iPSCs were differentiated to beating cardiomyocytes (iPSC-CMs),
and hypertrophic growth was induced via Endothelin-1 (ET-1) treatment together with siRNA
mediated silencing of FTO. I observed that cell growth was attenuated upon silencing of FTO
and expression of the stress-marker ANP was reduced.
To determine, how the demonstrated in-vitro effect presents in vivo, I bred and characterized
a cardiomyocyte specific Fto knockout (KO) mouse and applied the PO model to investigate
hypertrophy and heart failure in FTO-deficient mice. Intriguingly, KO mice show a maladaptive
response to PO, with early onset of dilatation and significantly impaired cardiac function
compared to control mice. Therefore, I hypothesize that the attenuated hypertrophy observed
in vitro represents the inability of FTO-depleted CMs to undergo initial compensatory
adaptation as seen in the in vivo model.
Together, my findings underline the importance of m6A RNA methylation in cardiac
hypertrophy and heart failure progression and provide insight into the manipulation of FTO as
a potential therapy in pressure overloaded hearts. | de |