In the present study 25 microRNAs were found to be dysregulated by pharmacological intervention with DOX in the rat heart. A subset of microRNA candidates were further investigated and associated to their putative targets.
The chronic weekly intravenous administration of DOX was designed to trigger cardiomyopathy symptoms within a 6 week time-frame allowing us to identify mRNAs and microRNAs that are altered in the early stages of the drug-induced cardiac remodeling. Cardiac toxicity at a cumulative dose of 12 mg/kg (3 mg/kg/week for 4 weeks) was confirmed by histopathological lesions and altered expression of genomic indicators associated to contraction performance (Myh6, Myh7) and heart failure (Ankrd1/CARP, Nppb) () 
. In most of the cases the dysregulation of these genes reached a significantly higher level between 2 and 4 weeks of the DOX 3 mg/kg/week dosing regimen. Additionally, 2 weeks treatment with DOX at 2 mg/kg/week onward caused cytoplasm vacuolation to occur with increasing severity.
We profiled hundreds of microRNAs via TLDAs in the heart of rats that showed clear signs of toxicity (DOX 3 mg/kg for 4 weeks), and found that 25 microRNAs were dysregulated with p values below 0.05. Seven out of 25 microRNAs (miR-367, miR-216b, miR-383, miR-692, miR-135b, miR-145*, miR-877, miR-434-5p and miR-337-5p), were already modulated after 2 weeks of DOX at 3 mg/kg/week (). Comparable changes for many of these microRNAs were confirmed in additional animals, and a subset of five microRNAs (miR-208b, miR-215, miR-216b, miR-367 and miR-34c) and were largely unaffected by the direct Top2 inhibitor etoposide (EPS) (). However DOX-induced up regulation of these microRNAs was not lessened by co-treatment with DZR, suggesting that these were not involved in the molecular pathways triggered by DZR.
It is noteworthy that miR-21 and miR-146a, previously linked to DOX-induced apoptosis in cardiomyocytes in vitro
or by single administration of a high dose of DOX in vivo
, were not among the microRNAs regulated by our chronic DOX dosing regime in Sprague Dawley rats. Similarly, miR-1 and miR-133, known to have alternative effects on oxidative stress-induced apoptosis in myocytes 
, the former being pro-apoptotic and the latter anti-apoptotic, were not altered in the present study. These discrepancies may derive from several key differences in the model systems and study designs used. Importantly, primary cells or cell lines treated in vitro
are not comparable to the whole cardiac tissue when exposed to metabolized systemic DOX and the work of Horie et al.
explores the acute effect of a single high dose of DOX, which contrasts to the chronic regimen administered here. Our present study describes global changes in microRNAs during chronic drug-induced cardiac toxicity, in a way that closely resembles the safety signals observed in a clinical setting.
In our study miR-215, part of the miR-192/miR-215 cluster, and miR-34c, involved in DNA damage mediated proliferation arrest 
, were up-regulated, consistent with the pharmacological effect of DOX. However miR-215 and miR-34c were not significantly up-regulated by EPS administration, suggesting that this compound may not trigger a p53 response under these experimental conditions.
Interestingly miR-215, up regulated by DOX in this study, was also linked to p53 mediated cell cycle arrest 
, consistent with the pharmacology of DOX and partially replicating expression changes of miR-34c upon treatment, thus strengthening the biological relevance of our findings and consistency with previously published mechanisms 
MicroRNA-208b, encoded from the intron 28 of rat Myh7, is associated to maintenance of myocardial performance together with 2 other myomirs, i.e. miR-208a/miR-499, which play a pivotal role in the myosin balance 
. Consistent with previous work, the DOX-induced expression of miR-208b parallels the modulation of expression of its host gene Myh7 (Figure S1B
) and occurs in parallel to the appearance of vacuolation (Table S3
). The concomitant decrease of miR-208a and Myh6 levels upon DOX treatment (Figure S1A
) further support the importance of the regulatory role of myomiRs-myosin interactions in the myosin switch, which is associated with progressive structural changes and pathological cardiac remodeling 
Of particular interest is the predictive potential of microRNAs as early tissue indicators of drug-induced cardiac lesions. The lowest dose of DOX tested in our study (1 mg/kg/week for 2 weeks) did not cause detectable tissue vacuolation (Table S2
). However, miR-216b was already up-regulated (>1.5 fold) under these conditions, and miR-367 concomitantly increased at the earliest time point (2 weeks) in the different treatment groups. Treatment with DOX at all doses and time points was associated with sustained up-regulation of these microRNAs, together with miR-208b and miR-215 at later time points. Further studies will be needed to investigate the potential of circulating microRNAs as indicators of DOX- induced cardiac injury.
In order to gain further insight into the cellular processes that may be modulated upon DOX-induced cardiac toxicity, high confidence target mRNAs of DOX-dysregulated microRNAs were identified by using the Microcosm prediction tool (Table S4
). In particular, only the genes that had an anti-correlated change of expression at the mRNA level vs.
the cognate microRNA were retained. The screening for putative targets of microRNAs in DOX treated animals, revealed that a number of DOX-responsive microRNAs may regulate mRNAs involved in cardiac tissue remodeling (data not shown), but we also speculated that the Sipa1 mRNA transcripts could be targeted by miR-34c directly. Sipa1 gene product is an activator of Rap GTPase and can influence proliferation rate and metastatic potential in various types of malignancies 
. Moreover cAMP-mediated Rap signaling, and its modulators, played a role in significant aspects of cardiovascular physiology, including cardiomyocytes contraction and hypertrophy 
. Importantly, Sipa1 mRNA showed an anti-correlated pattern with miR-34c in H9c2 cells treated with DOX 1 µM during 24 h (). Reporter assay experiments further support that Sipa1 mRNA is a target of the miR-34 family, in particular miR-34c (). In addition, our observations in H9c2 myoblast cell line suggest that miR-34c could be involved in the stimulation of the positive autophagy regulator Ambra1, also in presence of DOX. In fact, miR-34c over expression showed a statistically significant increase of the endogenous levels of Ambra1 mRNA (both with and without DOX). Vice versa transfection of a miR-34c-directed hairpin inhibitor showed a statistically significant decrease of Ambra1 mRNA level. However, the functional relevance of this finding in autophagy regulation and the exact mechanism of this regulation will have to be elucidated. Further studies, such as gain- or loss-of-function in vivo
investigations, will be required to elucidate the functional significance of altered Sipa1 expression for cardiac responses to DOX. Immunochemistry staining of autophagy markers like Ambra1 in heart tissues will also be required to elucidate the functional involvement of miR-215, miR-216b, miR-367, miR-208b and miR-34c in the DOX-induced autophagy process since they seem to be specifically associated to vacuoles appearance.
In conclusion, 25 microRNAs implicated in DOX-induced cardiac toxicity in vivo were identified. Among them, miR-216b, which was significantly regulated before overt toxicity, has the potential of a genomic indicator of cardiac toxicity.
Our study also provides the basis for understanding the global role of microRNAs in DOX-induced cardiac remodeling and toxicity, and provides evidence the direct miR-34c/Sipa1 functional interaction for the first time.