Zidovudine-Mediated Autophagy Inhibition Enhances Mitochondrial Toxicity in Muscle Cells

Abstract

Nucleoside reverse transcriptase inhibitors (NRTI), such as zidovudine (AZT), are constituents of HIV-1 therapy and are used for the prevention of mother-to-child transmission. Prolonged thymidine analogue exposure has been associated with mitochondrial toxicities to heart, liver, and skeletal muscle. We hypothesized that the thymidine analogue AZT might interfere with autophagy in myocytes, a lysosomal degradation pathway implicated in the regulation of mitochondrial recycling, cell survival, and the pathogenesis of myodegenerative diseases. The impact of AZT and lamivudine (3TC) on C2C12 myocyte autophagy was studied using various methods based on LC3-green fluorescent protein overexpression or LC3 staining in combination with Western blotting, flow cytometry, and confocal and electron microscopy. Lysosomal and mitochondrial functions were studied using appropriate staining for lysosomal mass, acidity, cathepsin activity, as well as mitochondrial mass and membrane potential in combination with flow cytometry and confocal microscopy. AZT, but not 3TC, exerted a significant dose- and time-dependent inhibitory effect on late stages of autophagosome maturation, which was reversible upon mTOR inhibition. Inhibition of late autophagy at therapeutic drug concentrations led to dysfunctional mitochondrial accumulation with membrane hyperpolarization and increased reactive oxygen species (ROS) generation and, ultimately, compromised cell viability. These AZT effects could be readily replicated by pharmacological and genetic inhibition of myocyte autophagy and, most importantly, could be rescued by pharmacological stimulation of autophagolysosomal biogenesis. Our data suggest that the thymidine analogue AZT inhibits autophagy in myocytes, which in turn leads to the accumulation of dysfunctional mitochondria with increased ROS generation and compromised cell viability. This novel mechanism could contribute to our understanding of the long-term side effects of antiviral agents.

Molecular Mechanisms of Cardiomyocyte Death in Drug-Induced Cardiotoxicity

“…Fas/Fas L was also involved in zidovudine-induced cardiomyocytes apoptosis (Purevjav et al, 2007). Besides the above effects, zidovudine was shown to inhibit autophagosome maturation and decrease autophagic flux, leading to mitochondrial membrane polarization and ROS accumulation (Lin et al, 2019).…”

Antiretroviral Drugs Impact Autophagy with Toxic Outcomes

“…ZDV and d4T appear to inhibit autophagy in most cell types examined. In two separate studies, one group characterized autophagy changes in C2C12 cells, mouse myoblasts [ 13 ], and in differentiated adipocytes from a mouse fibroblast cell line, 3T3-F442A [ 14 ], after treatment with concentrations of ZDV or d4T ranging from therapeutic maximum (C max, which is approximately 6 and 3 µM, respectively) to 5× and 30× C max , to determine the mechanisms driving myopathy and lipodystrophy in PLWH. They show that APG maturation was significantly inhibited in both cell types dose-dependently, importantly, at C max of ZDV or d4T.…”

“…They show that APG maturation was significantly inhibited in both cell types dose-dependently, importantly, at C max of ZDV or d4T. This was accompanied by hyperpolarization of mitochondrial membranes in the myocytes [ 13 ], accumulation of mitochondria and inhibition of lipid acquisition in the adipocytes [ 14 ], and increased ROS and decreased cell viability in both cell types [ 13 , 14 ]. This phenotype was recapitulated after pharmacologic and genetic inhibition of autophagy in the different cells.…”

“…Lamivudine (3TC) remains an important component of the antiretroviral arsenal, and can also be used for post-exposure prophylaxis to prevent HIV infection after potential exposure [ 42 ]. 3TC was used in the same studies of myocytes, adipocytes, and HepG2 and HUH7 hepatocytes described above for ZDV and d4T [ 13 , 14 , 15 ]. 3TC had no effect on mitochondria nor on autophagy in any of the cell types [ 13 , 14 , 15 ].…”

“…3TC was used in the same studies of myocytes, adipocytes, and HepG2 and HUH7 hepatocytes described above for ZDV and d4T [ 13 , 14 , 15 ]. 3TC had no effect on mitochondria nor on autophagy in any of the cell types [ 13 , 14 , 15 ]. However, the peak steady state amount of 3TC, which is approximately 8 µM, caused significant electron transport chain dysfunction in HUVEC with increased ROS and decreased ATP production, and increased the LC3-II/I ratio as well as Nix, suggesting increased mitophagy [ 19 ].…”