Anthracycline exposure alters future cardiac stress responses potentially driven by FOXK1

V. Sunder (Heidelberg)¹, D. Finke (Heidelberg)¹, B. Eksi (Heidelberg)², J. Brauer (Heidelberg)¹, N. Frey (Heidelberg)¹, L. H. Lehmann (Heidelberg)^1
1Universitätsklinikum Heidelberg Klinik für Innere Med. III, Kardiologie, Angiologie u. Pneumologie Heidelberg, Deutschland; ²Universitätsklinikum Heidelberg Institut für experimentelle Kardiologie Heidelberg, Deutschland

Anthracyclines are the most used and efficient chemotherapy drugs for the treatment of several cancers. Their use is limited by their cardiotoxicity which presents itself as dilatative cardiomyopathy often several years after exposure to anthracyclines and the mechanisms underlying this delayed response remain unclear. We hypothesized that exposure to anthracyclines increases cardiac susceptible to future adverse stress response. To test this, neonatal rat cardiomyocytes were treated with low doses of doxorubicin for 1 h followed by beta-adrenergic stimulation using Isoproterenol (Iso) 5 days later.  Gene expression analysis revealed that an initial exposure to doxorubicin leads to increased expression of the pathological marker nppb (Iso, fc=2.79; Doxo+Iso, fc=5.5 vs. control, p= 0.0241, <0.0001).
A two-hit model using Doxo+Iso in C57B6 mice revealed that mice receiving Doxo+Iso had a worse systolic function compared to mice receiving Doxo or Iso alone. Transcriptomic analyses unraveled genes dysregulated by Doxo+Iso but not by Doxo alone. A motif-discovery analysis revealed FOXK1 to be the transcriptional regulator of these genes which include DUSP4 and PDK2. 
Interestingly, immunostaining of NRVM’s suggests that FOXK1 might localize to the mitochondria in cardiomyocytes. Epigenetic analysis of the mitochondrial genome, using ATAC-seq from isolated mitochondria from hearts treated with doxorubicin we identified a previously unrecognized regulatory region in the MT-RNR2 gene with less accessibility upon doxorubicin exposure. An unbiased in vitro proteomic-interaction screen of this region again identified FOXK1 as an interacting protein, suggesting a role in direct regulation of mitochondrial function. 
Our data highlight FOXK1 as a potential mediator of long-term cardiac anthracycline mitotoxicity by direct regulation of both mitochondrial function and through its role as a nuclear transcription factor.