BACKGROUND: F1F0-ATP synthase (F1F0-ATPase) plays important roles in regulating 
mitochondrial function during hypoxia, but the effect of F1F0-ATPase defect on 
hypoxia/reoxygenation (H/RO) is unknown. The aim of this study was to 
investigate how mtDNA T8993G mutation (NARP)-induced inhibition of F1F0-ATPase 
modulates the H/RO-induced mitochondrial dysfunction. In addition, the potential 
for melatonin, a potent antioxidant with multiple mitochondrial protective 
properties, to protect NARP cells exposed to H/RO was assessed.
METHODS AND FINDINGS: NARP cybrids harboring 98% of mtDNA T8993G genes were 
established as an in vitro model for cells with F1F0-ATPase defect; their 
parental osteosarcoma 143B cells were studied for comparison. Treating the cells 
with H/RO using a hypoxic chamber resembles ischemia/reperfusion in vivo. NARP 
significantly enhanced apoptotic death upon H/RO detected by MTT assay and the 
trypan blue exclusion test of cell viability. Based on fluorescence 
probe-coupled laser scanning imaging microscopy, NARP significantly enhanced 
mitochondrial reactive oxygen species (mROS) formation and mitochondrial Ca(2+) 
(mCa(2+)) accumulation in response to H/RO, which augmented the depletion of 
cardiolipin, resulting in the retardation of mitochondrial movement. With 
stronger H/RO stress (either with longer reoxygenation duration, longer hypoxia 
duration, or administrating secondary oxidative stress following H/RO), NARP 
augmented H/RO-induced mROS formation to significantly depolarize mitochondrial 
membrane potential (ΔΨm), and enhance mCa(2+) accumulation and nitric oxide 
formation. Also, NARP augmented H/RO-induced mROS oxidized and depleted 
cardiolipin, thereby promoting permanent mitochondrial permeability transition, 
retarded mitochondrial movement, and enhanced apoptosis. Melatonin markedly 
reduced NARP-augmented H/RO-induced mROS formation and therefore significantly 
reduced mROS-mediated depolarization of ΔΨm and accumulation of mCa(2+), 
stabilized cardiolipin, and then improved mitochondrial movement and cell 
survival.
CONCLUSION: NARP-induced inhibition of F1F0-ATPase enhances mROS formation upon 
H/RO, which augments the depletion of cardiolipin and retardation of 
mitochondrial movement. Melatonin may have the potential to rescue patients with 
ischemia/reperfusion insults, even those associated with NARP symptoms.