Table 3.

Evaluation of mitochondrial permeability transition-dependent necrosis

Characteristics of MPT	Differences Between MPT-Dependent and Other Forms Of Cell Death	Methods to Detect Characteristics	References
Loss of ΔΨm	Opening of the MPT pore will dissipate the ΔΨm; however, other interventions can also induce loss of ΔΨm and therefore appropriate controls must be used.	1) Detection: assessment of using potentiometric fluorophores such as TMRE or JC-1 both in cultured myocytes and epicardially in isolate perfused hearts; 2) inhibitors: cyclosporine-A, bongkrekic acid, CypD depletion, positive controls: Ca2+, H2O2, ionomycin	9, 13, 35, 48, 82, 165, 166, 168, 176, 180, 183, 188, 190
Increased IMM permeability	Rapid increase in IMM permeability ≤1.5 kD, leading to swelling, separate from the increase in OMM permeability induced during intrinsic apoptosis.	1) Detection: measurement of swelling and CRC in isolated cardiac mitochondria and CRC and calcein CoCl2 in intact myocytes. Use of 2-DOG uptake or loss of mitochondrial NAD+ in vivo; 2) inhibitors: cyclosporine-A, bongkrekic acid, CypD depletion, positive controls: Ca2+, H2O2, ionomycin	9, 13, 35, 54, 81, 82, 165, 166, 176, 183, 188, 190, 207
Inhibition of mitochondrial ATP synthesis	By dissipating the ΔΨm, opening of the MPT pore inhibits ATP synthesis, leading to a depletion in adenine nucleotides.	1) Detection: measurement of mitochondrial ATP levels in vitro and in vivo using commercially available kits, measurement of ATP synthesis rates in isolated mitochondria; 2) inhibitors: cyclosporine-A, bongkrekic acid, CypD depletion, positive controls: Ca2+, H2O2, ionomycin	48, 53, 84, 227

CRC, Ca²⁺ retention capacity; CypD, cyclophilin-D; IMM, inner mitochondrial membrane; OMM, outer mitochondrial membrane; MPT, mitochondrial permeability transition; TMRE, tetramethylrhodamine ethyl ester.