Mitochondrial ATP production is necessary for activation of the extracellular-signal-regulated kinases during ischaemia/reperfusion in rat myocyte-derived H9c2 cells

Abstract

To search for the stimuli involved in activating the mitogen-activated protein kinases (MAPKs) during ischaemia and reperfusion, we simulated the event in a system in vitro conducive to continuous and non-invasive measurements of several major perturbations that occur at the time: O2 tension, mitochondrial respiration and energy status. Using H9c2 cells (a clonal line derived from rat heart), we found that activation of the extracellular signal-regulated MAPKs (ERKs) on reoxygenation was abolished if the mitochondria were inhibited prior to and during reoxygenation. Re-introduction of O2per se is therefore not sufficient to activate the ERKs. Recovery and maintenance of cellular ATP levels by mitochondrial respiration is necessary, although ATP recovery alone is not sufficient. ERK activation by H2O2, but not phorbol esters, was also sensitive to mitochondrial inhibition. Thus, reoxygenation and H2O2-mediated oxidative stress share a mechanism of ERK activation that is ATP- or mitochondrion-dependent, and this common feature suggests that the reoxygenation response is mediated by reactive oxygen species. A correlation between ERK activity and ATP levels was also found during the anoxic phase of ischaemia, an effect that was not due to substrate limitation for the kinases. Our results reveal the importance of cellular metabolism in ERK activation, and introduce ATP as a novel participant in the mechanisms underlying the ERK cascade.

Response of mitochondrial reactive oxygen species generation to steady-state oxygen tension: implications for hypoxic cell signaling

“…Thus an effect of a classical mitochondrial inhibitor on cellular ROS generation does not necessarily indicate mitochondria are the source of ROS; it merely suggests that some aspect of mitochondrial function is required for cellular ROS generation (2,46). In agreement with this, it was recently shown that the activation of ERK, previously attributed to mitochondrial ROS generation, actually depends more on mitochondrial ATP synthesis than it does on ROS (1,8). Furthermore, many classical mitochondrial inhibitors are known to affect other cellular proteins.…”

Practical guidelines for rigor and reproducibility in preclinical and clinical studies on cardioprotection

“…They also adopt a more oxidative metabolism, characterized by good coupling between glycolysis and mitochondrial respiration and indicative of higher energetic efficiency [336]. Different reports agree that cardiac-like H9c2 cells resemble more closely adult cardiomyocytes than HL-1 cells in terms of their metabolic signature, mitochondrial respiratory function and vulnerability to ischemia/reperfusion injury [2, 265, 314]. …”

Molecular and cellular mechanisms of cardiotoxicity.

“…These include the most often described alterations in ionic homeostasis such as changes in intracellular calcium concentrations, which occur in almost all examined exposures to environmental toxicants to date (11,12). Aberrant energy metabolism is another early response to environmental toxicants in the heart, resulting in decreased production and/or enhanced consumption of adenosine triphosphate (ATP) (13,14). Alterations in enzymatic reactions are often described in cardiac toxic responses (15), although we know very little about these alterations.…”