Abstract
The clinical use of anthracyclines in cancer therapy is limited by dose-dependent cardiotoxicity that involves cardiomyocyte injury and death. We have tested the hypothesis that anthracyclines affect protein degradation pathways in adult cardiomyocytes. To this aim, we assessed the effects of doxorubicin (Doxo) on apoptosis, autophagy and the proteasome/ubiquitin system in long-term cultured adult rat cardiomyocytes. Accumulation of poly-ubiquitinated proteins, increase of cathepsin-D-positive lysosomes and myofibrillar degradation were observed in Doxo-treated cardiomyocytes. Chymotrypsin-like activity of the proteasome was initially increased and then inhibited by Doxo over a time-course of 48 h. Proteasome 20S proteins were down-regulated by higher doses of Doxo. The expression of MURF-1, an ubiquitin-ligase specifically targeting myofibrillar proteins, was suppressed by Doxo at all concentrations measured. Microtubule-associated protein 1 light chain 3B (LC3)-positive punctae and both LC3-I and -II proteins were induced by Doxo in a dose-dependent manner, as confirmed by using lentiviral expression of green fluorescence protein bound to LC3 and live imaging. The lysosomotropic drug chloroquine led to autophagosome accumulation, which increased with concomitant Doxo treatment indicating enhanced autophagic flux. We conclude that Doxo causes a downregulation of the protein degradation machinery of cardiomyocytes with a resulting accumulation of poly-ubiquitinated proteins and autophagosomes. Although autophagy is initially stimulated as a compensatory response to cytotoxic stress, it is followed by apoptosis and necrosis at higher doses and longer exposure times. This mechanism might contribute to the late cardiotoxicity of anthracyclines by accelerated aging of the postmitotic adult cardiomyocytes and to the susceptibility of the aging heart to anthracycline cancer therapy.
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We thank Franziska Seifriz for excellent technical assistance and Daniel Ott for contributions to Fig. 4.
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This work was supported by a Swiss National Science Foundation grant to C.Z. (3100A0-120664) and research grants from Hoffmann La-Roche to T.M.S.
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Fig. S1
Cytoskeleton degradation and protein aggregates in adult rat ventricular myocytes (ARVM) treated with doxorubicin (Doxo) at 10 μM. a-a’’’ Three cells (bracketed as 1–3 in a) displaying various stages of cytoskeleton degradation after treatment with 10 μM Doxo for 48 h and immunostained for titin in the M-line region (a’) by using antibody m8, for a calpain-sensitive region of titin in the I-band by using antibody 9D10 (a’’) and for F-actin by using rhodamin-phalloidin (a’’’). b-b’’’ Single confocal optical section of ARVM treated with 10 μM Doxo for 48 h and immunostained for myomesin (b’) and polyubiquitinated proteins (b’’). (JPEG 1600 kb)
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Dimitrakis, P., Romay-Ogando, MI., Timolati, F. et al. Effects of doxorubicin cancer therapy on autophagy and the ubiquitin-proteasome system in long-term cultured adult rat cardiomyocytes. Cell Tissue Res 350, 361–372 (2012). https://doi.org/10.1007/s00441-012-1475-8
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DOI: https://doi.org/10.1007/s00441-012-1475-8