Early effects of doxorubicin in perfused heart: transcriptional profiling reveals inhibition of cellular stress response genes

Abstract

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.

Cardiac phosphoproteome reveals cell signaling events involved in doxorubicin cardiotoxicity

“…Some of such approaches were recently applied to anthracycline cardiotoxicity: transcriptomics [13][14][15], proteomics including toxico-and redox-proteomics [16][17][18][19], as well as metabolomics [20]. However, no attempt has been made so far to identify phosphorylation and dephosphorylation events linked to altered cell signaling by protein kinases and phosphatases.…”

“…In this study, we applied gel-based phosphoproteomics for non-biased identification of protein (de)phosphorylation events during DXR perfusion of the rat heart, a model already characterized by our previous studies on energy metabolism [22] and the cardiac transcriptome [14]. In vitro models of anthracycline cardiotoxicity, including the Langendorff perfused heart, are limited in the duration of drug exposure and reduce the complexity of the in vivo situation.…”

Inorganic Nitrate Therapy Improves Doxorubicin-Induced Cardiomyopathy

“…Notably, a role for oxidative stress is supported by studies in genetically modified mice indicating that deficiency of antioxidant enzymes such as glutathione peroxidase-1, an important hydrogen peroxidedegrading enzyme, increased susceptibility to DOXinduced cardiotoxicity (5), whereas overexpression of manganese superoxide dismutase, an essential antioxidant enzyme in mitochondria, prevented these adverse effects of DOX (6). In an alternative concept for the mechanism of DOX-induced cardiotoxicity, independent of oxidative stress, it was recently hypothesized that DOX-dependent modulation of the phosphoproteome, cardiac transcriptional reprogramming, and especially inactivation of adenosine monophosphate-activated protein kinase (AMPK) and the creatine kinase, which are 2 key kinases involved in cardiac energy metabolism (7,8), might play a causal role. Because many kinases and phosphatases, including AMPK, are activated or inhibited by reactive oxygen and nitrogen species, however, these 2 hypotheses should not necessarily be seen as antagonistic, as low levels of reactive oxygen species (below the possibility of detection) under long-term DOX therapy might lead to modifications in the phosphorylation pattern of these enzymes, modulating their kinase and phosphatase activity.…”

“…1), inhibition of this enzyme would result in a deleterious feedback mechanism. Considering the recent findings on increased susceptibility of ALDH-2-deficient mice to ischemic damage in experimental myocardial infarction and inactivation of AMPK by 4-hydroxynonenal (23), there seems to be an important link between the observations on DOX-induced AMPK inacti- Inorganic Nitrate, the Cheap Cardiovascular Therapy vation (7,8) and a feasible explanation for the highly protective effects of inorganic nitrate and nitrite in ischemia-reperfusion models. Zhu et al (9) provide clear evidence that inorganic nitrate prevents mitochondrial dysfunction and mitochondrial oxidative stress, which provides a mechanistic background to the beneficial effects of inorganic nitrate on DOX-induced cardiotoxicity observed in previous reports.…”

Cardiac Toxicity

“…Most often, mitochondrial toxicity was only retrospectively analyzed after clinical reports of unwanted side effects. Although ex vivo systems using isolated perfused hearts are suitable to detect mitochondrial toxicity [93,119], this setup is not capable of medium or high throughput and is therefore not compatible with early stages of drug development and toxicity testing. Most recently, in vitro test systems for mitochondrial function and dysfunction have been developed that can be used either with transformed cell lines, isolated mitochondria from animal tissues, or with primary or ES-cell-derived cardiomoyocytes [116,120,121].…”