Ischemic acidosis causes apoptosis in coronary endothelial cells through activation of caspase-12

Sanjeev Kumar, Ruhr-Universität Bochum, Germany
Sascha Kasseckert, University of Giessen, Germany
Sawa Kostin, Max-Planck-Institute for Heart and Lung Research, Germany
Yaser Abdallah, Aga Khan University
Claudia Schafer, University of Giessen, Germany
Alexander Kaminski, University of Rostock, Germany
H Peter Reusch, Ruhr-Universität Bochum, Germany
Hans Michael Piper, University of Giessen, Germany
Gustav Steinhoff, University of Rostock, Germany
Yury Ladilov, University of Rostock, Germany

This work was published before the author joined Aga Khan University.



Myocardial ischemia has been shown to induce apoptosis of endothelial cells (EC). However, the mechanism of this endothelial injury is still poorly understood. To analyse the signaling pathway of ischemia-induced EC apoptosis was the aim of the present study.


The primary culture of rat coronary EC was exposed to simulated ischemia (glucose-free anoxia at pHo 6.4). Apoptosis was defined by staining of nuclei with Hoechst-33342 and TUNEL. Cytosolic Ca2+ and pH were measured with Fura-2 and BCECF, respectively.


Apoptosis (29.2±1.7% of cells) induced by exposure to simulated ischemia for 2 h was accompanied by cytosolic Ca2+ overload (1090±52 nmol/l) and acidosis (pHi=6.52±0.13). Simulated ischemia had no significant effect on caspase-8 cleavage, but induced cleavage of caspase-3 and caspase-12 and led to a slight release of cytochrome C. Prevention of cytosolic acidosis (anoxia at pHo 7.4) had no effect on cytochrome C release, but significantly reduced apoptosis, attenuated cytosolic Ca2+ overload, and prevented cleavage of caspase-12. A similar effect was achieved by inhibition of Ca2+ release channels in the endoplasmic reticulum with ryanodine and xestospongin C. Knock-down of caspase-12 with small interfering RNA suppressed caspase-3 activation and reduced apoptotic cell number by about 70%.


Acidosis, rather than anoxia, is an important trigger of apoptosis in EC under simulated ischemia. The main pathway of the simulated ischemia-induced apoptosis consists of the Ca2+ leak from the ER followed by activation of caspase-12 and caspase-3.