Our results suggest that in the doxorubicin-challenged heart, a combined energetic, oxidative, and genotoxic stress elicits a specific, hierarchical response where AMPK is inhibited at least partially by the known negative cross-talk with Akt and MAPK pathways, largely triggered by DNA damage signalling. Although such signalling can be protective, e.g. by limiting apoptosis, it primarily induces a negative feedback that increases cellular energy deficits, and via activation of mTOR signalling, it also contributes to the pathological cardiac phenotype in chronic doxorubicin toxicity.

Doxorubicin (DXR) belongs to the most efficient anticancer drugs. However, its clinical application is limited by the risk of severe cardiac-specific toxicity, for which an efficient treatment is missing. Underlying molecular mechanisms are not sufficiently understood so far, but nonbiased, systemic approaches can yield new clues to develop targeted therapies. Here, we applied a genome-wide transcriptome analysis to determine the early cardiac response to DXR in a model characterized earlier, that is, rat heart perfusion with 2 muM DXR, leading to only mild cardiac dysfunction. Single-gene and gene set enrichment analysis of DNA microarrays yielded robust data on cardiac transcriptional reprogramming, including novel DXR-responsive pathways. Main characteristics of transcriptional reprogramming were 1) selective upregulation of individual genes or gene sets together with widespread downregulation of gene expression; 2) repression of numerous transcripts involved in cardiac stress response and stress signaling; 3) modulation of genes with cardiac remodeling capacity; 4) upregulation of "energy-related" pathways; and 5) similarities to the transcriptional response of cancer cells. Some early responses like the induction of glycolytic and Krebs cycle genes may have compensatory function. Only minor changes in the cardiac energy status or the respiratory activity of permeabilized cardiac fibers have been observed. Other responses potentially contribute to acute and also chronic toxicity, in particular, those in stress-responsive and cardiac remodeling transcripts. We propose that a blunted response to stress and reduced "danger signaling" is a prime component of toxic DXR action and can drive cardiac cells into pathology.

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Doxorubicin is one of the most powerful drugs used in chemotherapy of a large number of cancers. However, its anti-tumor effects are associated with serious cardiotoxicity, which can lead to heart failure. So far, mechanisms responsible for cardiotoxicity are not fully understood. Here we provide evidence that persistent alterations in protein kinase cell signaling may play a key role in the etiology of cardiotoxicity. In this study, we apply targeted analysis of key protein kinase pathways as well as non-biased analysis of the entire cardiac phosphoproteome in two different model systems: isolated perfused rat heart, and heart from doxorubicin-treated rats. Although doxorubicin induces energetic, oxidative and genotoxic stress in the heart, activity of the energy stress sensor AMP-activated protein kinase is paradoxically down-regulated. Pro-survival MAPK and Akt pathways are activated, the latter via DNA damage sensed by DNA-PK. This is at least partially responsible for low AMPK activity, since Akt inhibition can restore AMPK activation. Combined inhibition of AMPK and activation of Akt and MAPKs also leads to activation of growth-stimulating mTOR signaling. Such signalling increases cellular energy deficits and, via active mTOR signaling, also contributes to the pathological cardiac phenotype. Cardiac phosphoproteomics based on 2D-gels and mass spectrometry revealed further alterations of phosphorylation and dephosphorylation events that are associated with the early response to doxorubicin. Some candidate phosphoproteins with putative functions in cardiotoxicity are currently under investigation. This study emphasizes the importance of cell signaling for our understanding of doxorubicin cardiotoxicity

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