Temporal changes in mitochondrial function and reactive oxygen species generation during the development of replicative senescence in human fibroblasts

https://doi.org/10.1016/j.exger.2022.111866Get rights and content

Highlights

  • Mitochondrial dysfunction does not occur in the transition phase before senescence.

  • Excess ROS generation does not occur in the transition phase before senescence.

  • GDF15 expression through ISR is not induced during replicative senescence.

  • Mitochondria are elongated without aberrant cristae structures in senescent cells.

  • Mitophagy-related proteins decline during replicative senescence.

Abstract

Mitochondria are dysfunctional in post-senescent cells. Therefore, age-dependent impairment of mitochondrial energy production accompanied by excessive mitochondrial reactive oxygen species (ROS) is proposed to be a key driver of cellular senescence, which is a state of irreversible cell cycle arrest. However, it remains to be clarified whether mitochondrial dysfunction initiates or accelerates replicative senescence. In this study, we observed no increase in mitochondrial ROS or decrease in mitochondrial respiratory function in human TIG-1 fibroblasts in the transition phase, during which the population doubling rate gradually decreases due to the development of replicative senescence. The integrated stress response and expression of growth differentiation factor 15, which are triggered by respiratory chain deficiency, were also not induced in the transition phase. Mitochondria were elongated without aberrant cristae structures in the transition phase. Mitophagy-related protein levels started to decrease in the transition phase, but autophagic flux slightly increased during replicative senescence. These results suggest that mitochondrial dysfunction and excessive mitochondrial ROS generation do not occur predominately in the transition phase and may not play a role in the development of replicative senescence in normal diploid TIG-1 fibroblasts.

Introduction

The mitochondrial free radical theory of aging proposes that aging is accompanied by mitochondrial dysfunction, which leads to an increase in the level of reactive oxygen species (ROS) (Harman, 1965). Indeed, many reports suggest that there is an association between ROS and aging (Kirby et al., 2002; Melov et al., 2001; Ohsawa et al., 2008). The mitochondrial membrane potential (ΔΨm) decreases and the level of mitochondrial ROS increases in post-senescent cells (Estrada et al., 2013; Hutter et al., 2004; Passos et al., 2007), supporting the conclusion that mitochondrial dysfunction and excessive mitochondrial ROS are key drivers of cellular senescence (Chapman et al., 2019). Physiological generation of ROS by mitochondria occurs as a by-product of electron leakage from the electron transport chain during oxidative phosphorylation, and almost all ROS are reduced by the antioxidant defense system. The remaining ROS lead to continuous accumulation of oxidative DNA damage and, finally, senescence. Excessive mitochondrial ROS elicited by chemoptogenetic damage of mitochondria can diffuse to the nucleus and damage telomeres (Qian et al., 2019). Furthermore, manipulations that decrease mitochondrial ROS delay replicative senescence (Packer and Fuehr, 1977; Passos et al., 2007). However, there are conflicting reports regarding the effect of mitochondrial ROS on aging. One study reported that an increase in mitochondrial ROS does not accelerate aging in mice, while others showed that such an increase extends the lifespans of model organisms such as yeast and worms (Doonan et al., 2008; Mesquita et al., 2010; Van Raamsdonk and Hekimi, 2009). Therefore, the roles of mitochondrial dysfunction and excessive mitochondrial ROS in aging remain inconclusive.

Cellular senescence is a state of irreversible cell cycle arrest triggered by various types of cellular stress, environmental stimuli, and developmental signals. Accumulating evidence suggests that increased numbers of senescent cells in aged tissues are associated with aging and age-related diseases (Gorgoulis et al., 2019). In vitro studies revealed the features of senescent cells, including activation of the DNA damage response (DDR), alteration of intracellular metabolism, changes in gene expression, and development of the senescence-associated secretory phenotype (Gorgoulis et al., 2019). Replicative senescence, a seminal discovery of Hayflick and Moorhead, is a type of cellular senescence attributed to telomere attrition due to long-term cultivation of cells. Normal diploid fibroblasts from fetal tissues were used in studies of replicative senescence. Before entering permanent cell cycle arrest (senescence phase), these cells first proliferate vigorously (proliferation phase) and then proceed to the transition phase, during which the population doubling rate gradually decreases. Critical changes associated with the development of replicative senescence occur in the transition phase before cells reach permanent cell cycle arrest. However, it is unclear whether severe mitochondrial dysfunction accompanied by excessive ROS production is needed to induce cellular senescence. Indeed, mitochondrial respiratory function is maintained in senescent cells (Hutter et al., 2004; Kim et al., 2018; Nacarelli et al., 2019; Takebayashi et al., 2015), and normal mitochondria are required for the development of senescence-related phenotypes (Correia-Melo et al., 2016). These findings prompted us to reconsider the significance of mitochondrial dysfunction and excessive mitochondrial ROS in replicative senescence.

In the present study, we cultured TIG-1 cells, which are human fetal lung-derived normal diploid fibroblasts, until they reached replicative senescence, and defined their proliferation, transition, and senescence phases. In each phase, senescence markers and mitochondrial morphology and ultrastructure were analyzed. To clarify whether mitochondrial dysfunction and excessive mitochondrial ROS production occurred in the transition phase, we compared mitochondrial respiratory chain function and ROS levels among the three phases. We found that mitochondrial function did not decrease and the level of mitochondrial ROS did not increase in the transition phase.

Section snippets

Characterization of TIG-1 cells in the transition phase

TIG-1 human fetal lung fibroblasts are suitable for analysis of replicative senescence because they only replicate a few times when initially cultured. We cultured TIG-1 cells until they reached replicative senescence. The population doubling level (PDL) was around 30 when serial cultivation was started and increased to around 70 when cells entered replicative senescence (Fig. 1A). The PDL per passage began to progressively decrease when it fell below around 4 (Fig. S1). Therefore, we defined

Discussion

Oxidative stress occurs when the steady-state levels of ROS increase and surpass cellular catabolism or detoxification defenses, resulting in cell injury. Physiological levels of ROS accelerate telomere shortening rates and the rate of cellular senescence (von Zglinicki et al., 2000). In addition, excessive mitochondrial ROS are a critical mediator of cellular senescence (Passos et al., 2010; Passos et al., 2007; Qian et al., 2019). These findings raise the question of whether excessive ROS

Cell culture

TIG-1 cells at a PDL of 23 were obtained from the Japanese Collection of Research Bioresources cell bank. TIG-1 cells thawed for the first time were expanded by passaging 1–3 times and preserved in cryopreservation medium (CELLBANKER 1; Takara Bio, Inc., Shiga, Japan) at −80 °C. Serial cultivation of TIG-1 cells was started at a PDL of 28–33. To evaluate proliferation rates, thawed cells were seeded on 100 or 150 mm cell culture dishes (IWAKI, Shizuoka, Japan) and cultured in Eagle's minimum

CRediT authorship contribution statement

  • Yasunori Fujita: Conceptualization, Methodology, Investigation, Writing-Original draft.

  • Masumi Iketani: Resources, Writing-Review & editing.

  • Masafumi Ito: Writing-Review & editing.

  • Ikuroh Ohsawa: Conceptualization, Writing-Review & editing, Supervision.

Declaration of competing interest

The authors declare no competing interests.

Acknowledgments

This study was supported in part by the Japan Society for the Promotion of Science (JSPS), KAKENHI Grant Number JP20K11525 (to Y.F.).

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