Conclusions

To complement existing data on cardiac effects after BET inhibition I have analyzed the survival of JQ1-treated mice after TAC but could not find any significant difference to vehicle treated animals. Indeed, the previously described cardio-protective effects could not be reproduced in this work, possibly due to factors such as gender and genetic background of the examined mice. Therefore, further investigation is needed for clarification.

Next, to explore the potential of targeting BET proteins as treatment of HF and to overcome limitations of previous studies on BET inhibition, I investigated the loss of the first bromodomain

of Brd2 and the complete loss of Brd4 in cardiomyocytes in healthy mice as well as after induction of PO. The deletion of the first bromodomain of Brd2 did not elicit any substantial phenotype during development, in the adult heart, or after induction of pressure overload leading to the conclusion that the first bromodomain of BRD2 or the full length BRD2 is not essential in murine cardiomyocytes. These findings are relevant in regard to therapeutic BET inhibition, as directed inhibition of the first BET bromodomain in a non-cardiac context most probably would not affect cardiac cells in terms of BRD2 function.

However, a cardiomyocyte-specific knockout of Brd4 was lethal if induced during early embryonic development but a knockout during adolescence led to the development of a hypertrophic cardiomyopathy (HCM) with the animals surviving for over twelve months. In conclusion, BRD4 is essential during cardiac development and necessary for negative regulation of genes promoting cardiac remodeling and hypertrophy in differentiated cardiomyocytes. Furthermore, Brd4 KO mice showed an increased mortality within the first four weeks after TAC, which is most likely due to diastolic dysfunction, arrythmias, and sudden cardiac death common for HCM. Nevertheless, surviving Brd4 KO mice showed better heart function than TAC control animals, which might point towards some cardio-protective effect of cardiomyocyte-specific Brd4 depletion. In addition, cardiac stress had almost no impact on gene expression as only 34 DEGs were detected (padj<0.05, log2FC±0.5) for Brd4 KO mice after TAC compared to Sham (Figure 3.33C). A likely explanation based on previous reports of BRD4 being integral for the response to hemodynamic stress (Anand et al. 2013; Spiltoir et al.

2013; Haldar and McKinsey 2014) is that our Brd4 KO mice lose the ability to integrate such stress due to the absence of BRD4 from cardiomyocytes. A second, more speculative, explanation might be that a BRD4-independent response to TAC is somehow blunted or inhibited due to Brd4 KO mediated expression changes preceding the surgery such as the induction of HCM.

Based on the findings of this thesis complemented with relevant literature I propose two distinct cardiac functions for BRD4: first, the negative regulation of e.g. pro-hypertrophic genes in the healthy heart and, second, the activation of pro-hypertrophic and other pathologic genes after pressure overload (Figure 4.1). BRD4 has recently been proposed as co-repressor in non-cardiac tissues and cells (Conrad et al. 2017; Sakamaki et al. 2017; Lambert et al. 2018) but the underlying mechanisms remain elusive. The dual BRD4 function could be mediated by its interaction with negative transcriptional regulators such as EHMT2 (Sakamaki et al. 2017), NuRD (Lambert et al. 2018), SWI/SNF nucleosome remodelers (Conrad et al. 2017) or with positive transcriptional regulators like P-TEFb (Jang et al. 2005; Yang et al. 2005, p. 200). The functional shift could also be mediated by the switch from the short BRD4 to its long isoform after stress-induction as demonstrated in 3.1.2 hence enabling interaction with either an inactive or active P-TEFb complex (Figure 4.1) (Schröder et al. 2012).

Figure 4.1: A model of BRD4 function in cardiomyocytes including relevant literature.

BRD4 has possibly two distinct functions in cardiomyocytes of the adult murine heart. After pressure overload BRD4 and active P-TEFb (CDK9, CyclinT) induce transcription of the stress response gene program. Findings from this work suggest that in the healthy heart BRD4 co-represses target genes involved in energy metabolism, Ca²⁺ handling, cardiac muscle contraction, ECM coupling, and cell growth as the knockout of Brd4 leads to their induction. In its absence BRD4 target genes get probably activated by another chromatin reader or a different mechanism. The negative gene regulation might be mediated through chromatin occupation by BRD4 in interaction with e.g. an inactive P-TEFb (HEXIM1-7SK-bound) complex or other negative transcriptional regulators. However, the functional shift from transcriptional repression to activation upon pressure overload could be mediated by a stress-induced isoform switch from short to long BRD4, which prevents the association of the inhibitory HEXIM1-7SK complex to P-TEFb thus promoting the transcription of stress response genes.

Although the underlying mechanisms must be elucidated in future studies, my findings underscore the therapeutic potential of BRD4 for heart failure and depending on the condition might allow two distinct strategies. As the knockout of Brd4 induced HCM in healthy mice it would be interesting to test if e.g. an AAV-mediated overexpression of the short BRD4 isoform would rescue the phenotype in these mice. If successful, this approach could be tested in an independent HCM model. Furthermore, I concluded that the stress response might be mediated by the long BRD4 isoform. To challenge this hypothesis and to test if an isoform shift could be therapeutically exploited, the long but not the short BRD4 isoform could be silenced in mice that underwent TAC surgery.

Sarcomere