Doxorubicin impairs cardiomyocyte viability by suppressing transcription factor EB expression and disrupting autophagy

Abstract

Doxorubicin (DOX) is an effective anti-cancer agent. However, DOX treatment increases patient susceptibility to dilated cardiomyopathy. DOX predisposes cardiomyocytes to insult by suppressing mitochondrial energy metabolism, altering calcium flux, and disrupting proteolysis and proteostasis. Prior studies have assessed the role of macroautophagy in DOX cardiotoxicity; however, limited studies have examined whether DOX mediates cardiac injury through dysfunctions in inter- and/or intra-lysosomal signaling events. Lysosomal signaling and function is governed by transcription factor EB (TFEB). In the present study, we hypothesized that DOX caused myocyte injury by impairing lysosomal function and signaling through negative regulation of TFEB. Indeed, we found that DOX repressed cellular TFEB expression, which was associated with impaired cathepsin proteolytic activity across in vivo, ex vivo, and in vitro models of DOX cardiotoxicity. Furthermore, we observed that loss of TFEB was associated with reduction in macroautophagy protein expression, inhibition of autophagic flux, impairments in lysosomal cathepsin B activity, and activation of cell death. Restoration and/or activation of TFEB in DOX-treated cardiomyocytes prevented DOX-induced suppression of cathepsin B activity, reduced DOX-mediated reactive oxygen species (ROS) overproduction, attenuated activation of caspase-3, and improved cellular viability. Collectively, loss of TFEB inhibits lysosomal autophagy, rendering cardiomyocytes susceptible to DOX-induced proteotoxicity and injury. Our data reveal a novel mechanism wherein DOX primes cardiomyocytes for cell death by depleting cellular TFEB.

TFEB activation protects against cardiac proteotoxicity via increasing autophagic flux

“…For instance, cardiac glucolipotoxicity in obesity and diabetes has been linked to diminished TFEB and autophagic flux in cardiomyocytes [48]. Disrupting the ALP through impairing lysosome acidification and repressing TFEB signaling was recently reported to contribute to doxorubicin cardiotoxicity [49, 50]. TFEB-mediated transactivation of ALP machinery was proposed to mediate the protection by intermittent fasting against myocardial ischemia-reperfusion injury [28].…”

Validation of optimal reference genes for quantitative real time PCR in muscle and adipose tissue for obesity and diabetes research

“…Ventricular calcium-tolerant myocytes were prepared by a previously described procedure 52 . Cardiomyocytes were plated on laminin coated plates at a final cell density of 40–70 × 10 3 cells/plate, incubated at 37 °C.…”

Autophagy and mitophagy in the context of doxorubicin-induced cardiotoxicity

“…Immunofluorescence-based detection of ‘dots’ (autophagosomes) containing either endogenous LC3-II, or overexpressed GFP-LC3 also pointed to the upregulation of autophagosome formation or accumulation, in in vitro and in vivo studies [11, 68, 77, 79, 82, 85]. In some models, however, decreased relative levels of lipidated LC3-II in response to Dox were reported [86–88]. Dox induced up-regulation of Beclin-1 and Atg-5 has been corroborated by several studies [68, 77, 80].…”

“…There is an emerging consensus, strengthened by recent studies, that Dox inhibits autophagic flux, by inhibiting lysosomal biogenesis and/or lysosomal function [72, 75, 88, 105, 106]. Bartlett and colleagues demonstrated that Dox decreases expression of TFEB (transcription factor EB) which regulates the expression of genes related to autophagy and lysosomal biogenesis [88].…”