Mitochondrial calcium signalling and cell death: Approaches for assessing the role of mitochondrial Ca2+ uptake in apoptosis

Abstract

SummaryLocal Ca 2+ transfer between adjoining domains of the sarcoendoplasmic reticulum (ER/SR) and mitochondria allows ER/SR Ca 2+ release to activate mitochondrial Ca 2+ uptake and to evoke a matrix [Ca 2+ ] ([Ca 2+ ] m ) rise. [Ca 2+ ] m exerts control on several steps of energy metabolism to synchronize ATP generation with cell function. However, calcium signal propagation to the mitochondria may also ignite a cell death program through opening of the permeability transition pore (PTP). This occurs when the Ca 2+ release from the ER/SR is enhanced or is coincident with sensitization of the PTP. Recent studies have shown that several pro-apoptotic factors, including members of the Bcl-2 family proteins and reactive oxygen species (ROS) regulate the Ca 2+ sensitivity of both the Ca 2+ release channels in the ER and the PTP in the mitochondria. To test the relevance of the mitochondrial Ca 2+ accumulation in various apoptotic paradigms, methods are available for buffering of [Ca 2+ ], for dissipation of the driving force of the mitochondrial Ca 2+ uptake and for inhibition of the mitochondrial Ca 2+ transport mechanisms. However, in intact cells, the efficacy and the specificity of these approaches have to be established. Here we discuss mechanisms that recruit the mitochondrial calcium signal to a pro-apoptotic cascade and the approaches available for assessment of the relevance of the mitochondrial Ca 2+ handling in apoptosis. We also present a systematic evaluation of the effect of ruthenium red and Ru360, two inhibitors of mitochondrial Ca 2+ uptake on cytosolic [Ca 2+ Mitochondrial Ca 2+ transport mechanismsThe pathways of the mitochondrial Ca 2+ import and export are illustrated in Fig1. Ca 2+ traverses the outer mitochondrial membrane (OMM) primarily through the voltage dependent anion-selective channel (VDAC) [1][2][3]. The molecular nature of the proteins mediating the Ca 2+ transport across the inner mitochondrial membrane (IMM) remains unknown. The protein mediating Ca 2+ uptake is referred as the uniporter (UP) and has been identified as a Ca 2+ selective ion channel [4]. The UP passes Ca 2+ along the electrochemical gradient largely due Correspondence to: Dr. György Hajnóczky, Department of Pathology, Anatomy and Cell Biology, Suite 261 JAH, Thomas Jefferson University, Philadelphia PA 19107, USA, Tel.: (215) 503-1427, Fax.: (215) 923-2218, E-mail: E-mail: Gyorgy.Hajnoczky@jefferson.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. In the sequence from the opening of the IP3Rs to the opening of the PTP, the calcium signal propagation may be affec...

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