Mitochondrial Membrane Potential Drives Early Change in Mitochondrial Morphology After Acetaminophen Exposure

Abstract

Mitochondrial morphology plays a critical role in regulating mitochondrial and cellular function. It is well established that oxidative stress and mitochondrial injury are central to acetaminophen (APAP) hepatotoxicity. However, the role of mitochondrial dynamics, namely the remodeling of mitochondrial morphology through fusion and fission, has largely gone unexplored. To investigate this, we used primary mouse hepatocytes treated with APAP which allowed for real-time visualization of mitochondrial morphology using mitotracker green. We found that alterations in mitochondrial morphology were dose dependent, with a biphasic response in mitochondrial shape at higher APAP doses. Importantly, these two distinct mitochondrial morphologies corresponded with differences in mitochondrial respiratory function and polarization. The early change in mitochondrial morphology can be reversible and appears to be an adaptive response caused by alterations in membrane potential, which ultimately help preserve mitochondrial function. The later delayed change in mitochondrial morphology is irreversible and is driven by loss of mitochondrial membrane potential, decreased canonical fusion proteins, and alterations in mitochondrial lipid composition. Collectively, these later changes tilt the scales toward mitochondrial fission resulting in fragmented mitochondria with reduced functionality. This work provides evidence of adaptive early changes in mitochondrial morphology, which results in functional consequences that are dictated by the severity of APAP overdose.

Mitochondrial Dynamics in Drug-Induced Liver Injury

“…While general mitochondrial health and bioenergetic status can be derived through several techniques such as high content imaging of reactive oxygen species or measure-ment of mitochondrial respiration by instruments such as the Seahorse Analyzer, direct visual examination of changes in mitochondrial morphology typically requires microscopic techniques. Thus, mitochondrial dynamics have been predominantly evaluated in in vitro cell culture using fluorescence microscopy or high resolution confocal microscopy with mitochondrial-targeted dyes [45,46]. Additional techniques include live-cell reporter strategies to simultaneously monitor mitochondrial biogenesis and morphology using mitochondrial-targeted GFP expression under control of Nrf1 [47] as well as a 2D confocal imaging-based approach using Mitotracker ® staining that combines automatic mitochondrial morphology and dynamics analysis with fractal analysis in live small cell lung cancer (SCLC) cells [48].…”

“…Hepatocytes play a critical role in systemic metabolism and hence are among the cells which have the highest number of mitochondria. Mitochondrial dynamics have been studied in perfused liver as well as isolated primary rat [53] and mouse hepatocytes [45]. Though discrete globular or short tubular mitochondria were evident in hepatocytes [45], few fusion events and little movement activity was noted [53], indicating that normal hepatocytes do not undergo rapid alterations in mitochondrial dynamics under physiological conditions.…”

“…Mitochondrial dynamics have been studied in perfused liver as well as isolated primary rat [53] and mouse hepatocytes [45]. Though discrete globular or short tubular mitochondria were evident in hepatocytes [45], few fusion events and little movement activity was noted [53], indicating that normal hepatocytes do not undergo rapid alterations in mitochondrial dynamics under physiological conditions. The caveat, however, is that these experiments were carried out in isolated primary hepatocytes under in vitro cell culture conditions where cells dedifferentiate, and mitochondrial dynamics in hepatocytes in vivo could be more active.…”

“…This mitochondria toxicity, in part, is mediated by the phosphorylation [70] and subsequent translocation of c-Jun N-terminal kinase (JNK) to the mitochondria [71]. The role of mitochondrial dynamics in APAP-induced hepatotoxicity was initially described when elevations in Drp-1 and mitochondrial fission were identified after an APAP overdose in mice in vivo as well as in isolated mouse primary hepatocytes [45,72]. Further mechanistic investigation revealed a biphasic process whereby APAP exposure to primary mouse hepatocytes induced spheroid-shaped mitochondria, which could be reversed, before progressing towards pathological, fragmented mitochondria that was irreversible.…”

“…Further mechanistic investigation revealed a biphasic process whereby APAP exposure to primary mouse hepatocytes induced spheroid-shaped mitochondria, which could be reversed, before progressing towards pathological, fragmented mitochondria that was irreversible. The early change in mitochondrial morphology was caused by alterations in membrane potential, while the latter pathological change was due to decreased levels of mitochondrial fusion proteins and loss of mitochondrial phosphatidic acid [45], which can restrict DRP1mediated fission [40]. Moreover, pharmacological inhibition of JNK phosphorylation after APAP exposure prevented the degradation of Mfn2 [45], which can be targeted by JNK for proteasomal degradation [73].…”