| dc.description.abstracteng | Heart failure (HF) is the pathologic inability of the heart to supply the body with sufficient amounts of oxygen-rich blood. This increasingly common, life-threatening condition occurs in the final stage of various cardiac pathologies that reduce heart function. Common pharmacotherapies of HF aim to inhibit the renin-angiotensin system and adrenergic receptors that are activated in response to the reduced pumping function and have been available for over 20 years. However, the morbidity and mortality rates of affected patients remain high. To this day the development of new, more effective therapies poses a major challenge in medical research.
The new therapeutic strategy investigated in this work is based on increasing evidence that epigenetics play an important role in the pathogenesis of HF. The rationale behind targeting epigenetic processes to treat the development of HF is that they modulate multiple transcriptional networks simultaneously. For instance, the small molecule JQ1 was shown to displace the bromodomain and extraterminal domain (BET) reader proteins BRD2, BRD3, and BRD4 from chromatin, preventing re-expression of the fetal gene program, pathologic hypertrophy, and fibrosis after pressure overload (PO). To allow effective and safe application of BET inhibition as treatment for HF, it is necessary to assess if targeting BET proteins has added benefits in comparison to current pharmacotherapies such as improved survival and to elucidate functions of individual BET members specifically in cardiac cells. However, previous studies miss to report mortality rates for JQ1 treated animals and do not consider that JQ1 acts systemically and inhibits all four BET family members alike. Thus, the mechanisms underlying BET-mediated cardio protection remain elusive.
First, to characterize and validate cardiac BET expression at baseline and in response to PO I performed gene expression analysis and immunoblotting using hearts of adult wildtype mice. I identified Brd2 as the highest expressed BET family member in the heart with four times higher mRNA levels compared to Brd3/Brd4 and revealed TAC-induced expression of the long BRD4 isoform.
Second, to describe the effect of BET inhibition on life expectancy after PO induction I monitored the survival of JQ1-treated wildtype mice for up to 2 months after TAC and analyzed the hearts using echocardiography as well as histological and molecular methods. I found PO-dependent mortality unchanged with JQ1-mediated BET inhibition and observed pathologic changes such as expression of cardiac stress markers, cardiomegaly, cardiomyocyte hypertrophy, interstitial fibrosis, and systolic dysfunction, which were comparable to vehicle-treated animals after TAC. This contradicts previous reports on cardio-protective features of JQ1 in a mouse PO model (Anand et al. 2013). As experimental differences such as sex, age, mouse strain, TAC-performance, and JQ1-batch cannot be excluded as explanations for the discrepant results, future studies should take these possible confounding factors into account. Moreover, a reliable cardiac-specific biomarker of BET-inhibition should be explored to allow successful therapy monitoring.
Third, using conditional alleles I generated mice expressing a truncated BRD2 protein lacking the first bromodomain, Brd2∆BDI, and mice with a cardiomyocyte-specific Brd4-knockout, Brd4 KO, to investigate whether these gene deletions alter the response to PO. Homozygous Brd2∆BDI mice were viable and their hearts and cardiac functions were not significantly different from Cre-positive control mice at baseline and after PO induction. In contrast, cardiomyocyte-specific homozygous deletion of Brd4 during early embryonic development was lethal suggesting that BRD4 is essential during cardiogenesis. For further examination Brd4 KO was induced at postnatal week five and resulted in animals that were viable for over 12 months. Adult Brd4 KO mice showed basal concentric hypertrophy, preserved ejection fraction, mild interstitial fibrosis, and cardiac stress marker expression. These features are characteristic for hypertrophic cardiomyopathy (HCM) and were further supported by transcriptome analysis that revealed differential expression of genes involved in extracellular matrix remodeling, energy metabolism, sarcomere composition, and cardiac muscle contraction. Moreover, Brd4 KO mice subjected to TAC showed significantly higher mortality within the first month after surgery, which might be attributed to diastolic dysfunction or arrythmias. Nevertheless, no significant wall thickening or left ventricular mass increase, despite the basal hypertrophy was observed in Brd4 KO mice after TAC. This lack of stress response was confirmed by mRNA sequencing as no relevant changes were detected in Brd4 KO animals after TAC compared to Sham. However, Brd4 KO mice that survived the acute phase of PO showed better heart function in comparison to TAC control.
My findings suggest that the function of BRD2 in cardiomyocytes is either independent from its first bromodomain, substituted by another protein upon disruption, or not essential and therefore needs further investigation. Furthermore, beside the established function of BRD4 as co-activator of cardiac stress response, my findings lead to the conclusion that BRD4 has a second function as co-repressor of e.g. pro-hypertrophic genes in the healthy heart. I further propose that the shift between both cardiac functions might be mediated by a stress-induced switch from the short to the long Brd4 isoform and a respective interaction with e.g. an inactive or active P-TEFb complex (Schröder et al. 2012).
My thesis provides the first functional insight into cardiomyocyte-specific loss of Brd4 in vivo, links it to the development of HCM, establishes basal BRD4-mediated negative regulation of transcription, and provides evidence for its depletion to blunt stress-response. These findings could contribute to the development of more selective therapeutic approaches for HF as compared to inhibition of all BET members and to our understanding of HCM development and manifestation. | de |