Transcription Factor GATA4 Inhibits Doxorubicin-induced Autophagy and Cardiomyocyte Death

Abstract

Doxorubicin (DOX) is a potent anti-tumor drug known to cause heart failure. The transcription factor GATA4 antagonizes DOX-induced cardiotoxicity. However, the protective mechanism remains obscure. Autophagy is the primary cellular pathway for lysosomal degradation of long-lived proteins and organelles, and its activation could be either protective or detrimental depending on specific pathophysiological conditions. Here we investigated the ability of GATA4 to inhibit autophagy as a potential mechanism underlying its protection against DOX toxicity in cultured neonatal rat cardiomyocytes. DOX markedly increased autophagic flux in cardiomyocytes as indicated by the difference in protein levels of LC3-II (microtubule-associated protein light chain 3 form 2) or numbers of autophagic vacuoles in the absence and presence of the lysosomal inhibitor bafilomycin A1. DOX-induced cardiomyocyte death determined by multiple assays was aggravated by a drug or genetic approach that activates autophagy, but it was attenuated by manipulations that inhibit autophagy, suggesting that autophagy contributes to DOX cardiotoxicity. DOX treatment depleted GATA4 protein levels, which predisposed cardiomyocytes to DOX toxicity. Indeed, GATA4 gene silencing triggered autophagy that rendered DOX more toxic, whereas GATA4 overexpression inhibited DOX-induced autophagy, reducing cardiomyocyte death. Mechanistically, GATA4 up-regulated gene expression of the survival factor Bcl2 and suppressed DOX-induced activation of autophagy-related genes, which may likely be responsible for the anti-apoptotic and anti-autophagic effects of GATA4. Together, these findings suggest that activation of autophagy mediates DOX cardiotoxicity, and preservation of GATA4 attenuates DOX cardiotoxicity by inhibiting autophagy through modulation of the expression of Bcl2 and autophagy-related genes. Doxorubicin (DOX)4 is a very effective anti-cancer drug with cardiotoxicity that culminates in congestive heart failure (1-3).The prevailing mechanism for DOX cardiotoxicity is oxidative stress that has been supported by the ability of several antioxidants to reduce DOX cardiotoxicity in animal studies (4 -8). Unfortunately, clinical trials have failed to reproduce these results in humans (9), suggesting that mechanisms other than oxidative stress might also contribute to DOX-induced heart failure (9 -13). In support of this notion, DOX has been shown to cause cardiac mitochondrial injury by both oxidative and non-oxidative mechanisms (14).Autophagy is the major cellular pathway for degrading and recycling long-lived proteins and organelles that are sequestered in double-membrane vesicles termed autophagosomes. After fusing with the lysosome to form autolysosome, the inner membrane and the contents are degraded and recycled. More than 30 autophagy-related (ATG) genes encode proteins essential for autophagy induction and the generation, maturation, and recycling of autophagosomes or autophagic vacuoles (15,16). Autophagy has long been recognized to provide a survival pathw...

Curcumin Attenuates Doxorubicin-Induced Cardiotoxicity by Inducing Autophagy via the Regulation of JNK Phosphorylation

“…Sishi et al reported that upregulation of autophagy protected against DOXinduced cardiotoxicity [16]. In contrast, DOX-induced cardiomyocyte death was aggravated by the activation of autophagy but attenuated by the inhibition of autophagy through GATA4 gene silencing, suggesting that excessive autophagy contributes to DOX-induced cardiotoxicity [35]. Thus, the role of autophagy in DOX-induced cardiotoxicity is still controversial because autophagy plays roles in both cell protection and cell death.…”

Signalling mechanisms underlying doxorubicin and Nox2 NADPH oxidase‐induced cardiomyopathy: involvement of mitofusin‐2

“…In promoting cell death, oxidative stress from ROS, including superoxide and peroxynitrite, cause activation of kinase pathways (MAPK 4/7, checkpoint kinase 2, stress‐activated protein kinase, JNK). Suppression of transcription factors, GATA‐4 (Kobayashi et al ., 2010; Suzuki, 2011) and p300 (Poizat et al, 2005), is also linked to regulation of cell survival. Induction of small heat shock proteins (e.g.…”

Suppression of autophagy is protective in high glucose-induced cardiomyocyte injury

“…Adenoviruses expressing β-galactosidase (Ad β-gal), human BECN1 (AdBCN1), the shRNA targeting rat BECN1 mRNA (AdshBCN1), and control shRNA (AdshCon) were created as previously described. 68 Adenovirus expressing mTOR and adenovirus encoding mRFP-GFP tandem fluorescent-tagged LC3 (AdtfLC3) were the gifts of Dr. Chris Rhodes 56 and Dr. Junichi Sadoshima, 46 respectively. To generate adenoviral vector expressing ATG7, an insert containing Atg7 cDNA sequence was amplified by PCR from mycand flag-tagged ORF clone of Mus musculus Atg7 (OriGene, MR210108) and integrated into the TA cloning vector (Invitrogen, KNM2000-01).…”

“…Protein extracts from cultured cardiomyocytes were prepared as described previously, except that 1% of protease inhibitor cocktail (Sigma, P8340) was added to the extraction buffer. 68 Protein samples were subjected to PAGE, transferred to polyvinylidene difluoride membrane (Amersham Pharmacia, RPN3031 or Bio-Rad, 162-0177), and then blocked in 5% milk prepared with 0.1% Tween-20 Tris-buffered saline (TBST) solution for 1 h at room temperature. Primary antibodies were incubated overnight at 4°C in 2.5% milk.…”