Non-inferiority Test for a Continuous Variable with a Flexible Margin in an Active Controlled Trial: an Application to the “stratall ANRS 12110 / ESTHER” Trial

Reactive oxygen species arising from ischemia/reperfusion (I/R) cause damage to cardiac tissue. We examined the effects of mitochondrial phospholipid hydroperoxide glutathione peroxidase (mPHGPx) and cytosolic PHGPx (cPHGPx) overexpression on protection against simulated I/R in neonatal rat cardiac myocytes (NCM). Additionally, a protective combinatorial effect with heat shock proteins 60 and 10 (HSP60/10) was investigated. NCM were infected with adenoviral vectors expressing mPHGPx, cPHGPx, HSP60/10, or an empty control (Adv-) and submitted to 8 h of ischemia followed by 16 h of reoxygenation. mPHGPx infection led to a 40% decrease in malondialdehyde and 4-hydroxy-2(E)-nonenal following I/R (p < .05). Creatine kinase and lactate dehydrogenase release were decreased in both mPHGPx-infected and HSP60/10-infected cells (p < .05). The combination of mPHGPx and HSP60/10 overexpression led to further protection (p < .01). DNA laddering and histone-associated DNA fragments were decreased in PHGPx- and HSP60/10-infected cells (p < .01). Cytochrome c release from mitochondria was decreased in mPHGPx-infected cells. Furthermore, mPHGPx overexpression preserved electron transport chain complex IV function following simulated I/R (p < .05). These results indicate that overexpression of PHGPx provides protection against damage resulting from simulated I/R injury, particularly in the mitochondria, and that the combination of mPHGPx and HSP60/10 imparts an added protective effect.