AIMS: Opening of the mitochondrial permeability transition pore (mPTP) is an 
important step on the pathway towards cardiomyocyte death, defining the extent 
of injury following cardiac ischaemia and reperfusion. In isolated mitochondria, 
mPTP opening is triggered by calcium overload facilitated by oxidative stress. 
In isolated cells, however, it has been suggested that mPTP opening occurs 
before calcium overload and is stimulated by oxidative stress. Our objective was 
to establish the events that cause mPTP opening in the intact heart.
METHODS AND RESULTS: We performed multiphoton imaging of Langendorff-perfused 
mouse hearts expressing an inducible, Ca(2+)-sensitive reporter (circularly 
Permuted GFP and calmodulin (CaM), version 2), to examine the spatiotemporal 
relationship between [Ca(2+)](c), redox state, and mPTP opening in the intact 
heart during hypoxia and reoxygenation at sub-myocyte resolution. We found that 
during reperfusion, calcium waves propagated across multiple cells at 3.3 µm/s. 
mPTP opening caused an abrupt loss of mitochondrial membrane potential, measured 
using a potentiometric dye, which was invariably preceded by a rise in 
[Ca(2+)](c). The probability that localized [Ca(2+)](c) waves led to mPTP 
opening was greater early during reoxygenation. During reoxygenation, 
coordinated redox changes also occurred across large regions and preceded mPTP 
opening on average by 122 ± 38 s. Fewer [Ca(2+)] waves led to mPTP opening in 
the presence of mPTP inhibitor cyclosporin A or mitochondrial-targeted scavenger 
of reactive oxygen species, MitoQ.
CONCLUSION: These experiments define the spatiotemporal relationship between 
changes in [Ca(2+)](c), redox state and mPTP opening during reoxygenation in the 
intact heart. Tissue oxidation coincident with localized calcium waves together 
conspire to cause mPTP opening and subsequent cell death.