Progressive apoptotic cell death triggered by transient oxidative insult in H9c2 rat ventricular cells: a novel pattern of apoptosis and the mechanisms

Abstract

Many pathophysiological processes are associated with oxidative stress and progressive cell death. Oxidative stress is an apoptotic inducer that is known to cause rapid cell death. Here we show that a brief oxidative insult (5-min exposure to 400 microM H(2)O(2)), although it did not kill H9c2 rat ventricular cells during the exposure, triggered an intracellular death cascade leading to delayed time-dependent cell death starting from 1 h after the insult had been withdrawn, and this post-H(2)O(2) cell death cumulated gradually, reaching a maximum level 8 h after H(2)O(2) withdrawal. By comparison, sustained exposure to H(2)O(2) caused complete cell death within a narrow time frame (2 h). The time-dependent post-H(2)O(2) cell death was typical of apoptosis, both morphologically (cell shrinkage and nuclear condensation) and biochemically (DNA fragmentation, extracellular exposure of phosphatidylserines, and caspase-3 activation). A dichlorofluorescein fluorescent signal showed a time-dependent endogenous increase of reactive oxygen species (ROS) production, which was almost abolished by inhibition of the mitochondrial electron transport chain. Application of antioxidants (vitamin E or DTT) before H(2)O(2) addition or after H(2)O(2) withdrawal prevented the H(2)O(2)-triggered progressive ROS production and apoptosis. Sequential appearance of events associated with activation of the mitochondrial death pathway was found, including progressive dissipation of mitochondrial membrane potential, cytochrome c release, and late activation of caspase-3. In conclusion, transient oxidative stress triggers an intrinsic program leading to self-sustained apoptosis in H9c2 cells via cumulative production of mitochondrial ROS and subsequent activation of the mitochondrial death pathway. This pattern of apoptosis may contribute to the progressive and long-lasting cell loss in some degenerative diseases.

A cell-microelectronic sensing technique for the screening of cytoprotective compounds

“…Cell index of the absolute control cells that did not receive H 2 O 2 treatment remained constant for 20 h, but began to decline thereafter, probably due to the overgrowth of culture in the well. Vehicle-control cells (which received H 2 O 2 and vehicle, but no treatment with cytoprotective compounds), showed a rapid decline of cell index, presumably as a consequence of cell shrinkage after oxidative stress (20) that is detected by the real-time cell microelectronic sensing system. At later time points, inhibition of cell viability, proliferation and death are also likely to contribute to the decline in cell index.…”

Effect of Hypoxia Mimetic Cobalt Chloride on the Expression of Extracellular-Superoxide Dismutase in Retinal Pericytes

“…19) On the other hand, ROS has been reported to activate caspase-3 and promote apoptotic cell death. 20) We observed the activation of caspase-3 and DNA fragmentation by CoCl 2 treatment, and these phenomena were significantly inhibited by pre-treatment with NAC (Figs. 3A, B).…”

Vitamin D in Oxidative Stress and Diseases

“…Vitamin D 3 along with its metabolites influences directly or indirectly almost all metabolic processes such as proliferation, differentiation, apoptosis, inflammatory processes, and mutagenesis. Such multifactorial effects of vitamin D 3 can be a profitable source of new therapeutic solutions for two radically divergent diseases as cancer and neurodegeneration [134]. The discussed results could be relevant in the light of the use of vitamin D 3 to promote supplementation or to adjust therapeutic strategies in neurodegenerative disorders.…”

“…Recent investigations on cardiac myocytes showed that ROS produced by mitochondrial and oxidative stress can cause multiple changes in the cell structure and the function that are associated with a failing heart [133] and apoptosis [134] or autophagy [135]. On the other hand, ROS, along with NO, show antiapoptotic effects involving several signaling pathways; for example, not only activates proapoptotic signals [135] but also potently induces autophagy [136].…”