A mitochondrial-targeted cyclosporin A with high binding affinity for cyclophilin D yields improved cytoprotection of cardiomyocytes.

Mitochondrial CyP-D (cyclophilin-D) catalyses formation of the PT (permeability transition) pore, a key lesion in the pathogenesis of I/R (ischaemia/reperfusion) injury. There is evidence [Malouitre, Dube, Selwood and Crompton (2010) Biochem. J.

Mitochondrial targeting of cyclosporin A enables selective inhibition of cyclophilin-D and enhanced cytoprotection after glucose and oxygen deprivation.

CsA (cyclosporin A) is a hydrophobic undecapeptide that inhibits CyPs (cyclophilins), a family of PPIases (peptidylprolyl cis-trans isomerases). In some experimental models, CsA offers partial protection against lethal cell injury brought about by transient ischaemia; this is believed to reflect inhibition of CyP-D, a mitochondrial isoform that facilitates formation of the permeability transition pore in the mitochondrial inner membrane. To evaluate this further, we have targeted CsA to mitochondria so that it becomes selective for CyP-D in cells.

Evaluating cytochrome c diffusion in the intermembrane spaces of mitochondria during cytochrome c release.

Truncated Bid (tBid) releases cytochrome c from mitochondria by inducing Bak (and Bax) pore formation in the outer membrane. An important issue is whether a second tBid action, independent of Bak and Bax, is also required to enhance cytochrome c mobility in the intermembrane spaces. To investigate this, we developed a kinetic analysis enabling changes in the diffusibility of cytochrome c in the intermembrane spaces of isolated mitochondria to be differentiated from changes resulting from Bak activation.

Bax translocates to mitochondria of heart cells during simulated ischaemia: involvement of AMP-activated and p38 mitogen-activated protein kinases.

The cytosolic protein Bax plays a key role in apoptosis by migrating to mitochondria and releasing proapoptotic proteins from the mitochondrial intermembrane space. The present study investigates the movement of Bax in isolated rat neonatal cardiomyocytes subjected to simulated ischaemia (minus glucose, plus cyanide), using green fluorescent protein-tagged Bax as a means of imaging Bax movements. Simulated ischaemia induced Bax translocation from the cytosol to mitochondria, commencing within 20 min of simulated ischaemia and progressing for several hours.

Cyclophilin-D promotes the mitochondrial permeability transition but has opposite effects on apoptosis and necrosis.

Cyclophilin-D is a peptidylprolyl cis-trans isomerase of the mitochondrial matrix. It is involved in mitochondrial permeability transition, in which the adenine nucleotide translocase of the inner membrane is transformed from an antiporter to a non-selective pore. The permeability transition has been widely considered as a mechanism in both apoptosis and necrosis.

On the involvement of mitochondrial intermembrane junctional complexes in apoptosis.

The voltage dependent anion channel and the adenine nucleotide translocase are the principal proteins found in the mitochondrial outer and inner membranes, respectively. The two proteins can associate to form a junctional complex that establishes contact sites between the two membranes. This complex in turn recruits a range of proteins depending on the function to be executed.

Increased susceptibility of striatal mitochondria to calcium-induced permeability transition.

Mitochondria were simultaneously isolated from striatum and cortex of adult rats and compared in functional assays for their sensitivity to calcium activation of the permeability transition. Striatal mitochondria showed an increased dose-dependent sensitivity to Ca2+ compared with cortical mitochondria, as measured by mitochondrial depolarization, swelling, Ca2+ uptake, reactive oxygen species production, and respiration. Ratios of ATP to ADP were lower in striatal mitochondria exposed to calcium despite equal amounts of ADP and ATP under respiring and nonrespiring conditions.

Biphasic translocation of Bax to mitochondria.

Using green fluorescent protein-tagged Bax, we demonstrate that Bax is sequestered from the cytosol of cardiomyocytes in two distinct phases following the induction of apoptosis with staurosporine. In the first phase, lasting several hours, Bax removal from the cytosol was relatively small. In the second phase, Bax was very largely removed from the cytosol and sequestered into large aggregates associated with the mitochondria.

Mitochondrial intermembrane junctional complexes and their involvement in cell death.

Mitochondria establish contact sites between the inner and outer membranes. The contact sites are held together by junctional complexes of the adenine nucleotide translocase (ANT; inner membrane) and the voltage-dependent anion channel (VDAC; outer membrane). The junctional complexes act as multifunctional recruitment centres, binding a range of proteins according to the function to be executed.

Cyclophilin-A is involved in excitotoxin-induced caspase activation in rat neuronal B50 cells.

Glutamate and the NO donor, nitroprusside, synergistically induced the death of B50 cells from a rat CNS-derived neuroblastoma cell line. With low [nitroprusside] (10 microM) both nitroprusside and glutamate were required. Under these conditions, nuclei became pyknotic and caspases were activated.