*Purpose: Aged livers are far more susceptible to ischemia/reperfusion (I/R) injury than young livers. No therapeutic strategies are available to reduce I/R injury in elderly patients. Uncontrolled activation of calpains degrades substrates essential to cell survival, leading to irreversible tissue injury. Mitophagy is an evolutionarily conserved process that selectively clears dysfunctional mitochondria. Calpain activation and consequent defective mitophagy are key pathological events contributing to liver failure following I/R. Calpastatin (CAST) is an endogenous calpain inhibitor. How CAST affects mitophagy and mitochondria in aged livers is unknown.

*Methods: To determine the age-dependent changes in CAST after ischemia in human livers, non-tumorous specimens were collected from the patients at different ages who underwent liver resection. Liver specimens were analyzed before and after 15 min of one episode of inflow occlusion. For studying mechanistic insights, livers or isolated hepatocytes from young (3-month-old) and aged (26-month-old) male C57 BL/6J mice were subjected to mild in vivo or in vitro ischemia by clamping the portal triad for 30 min or incubating in the anoxic medium for 2 h, respectively, conditions that minimally injure young livers and hepatocytes. Some cells were treated with adenoviral vector encoding CAST (Ad-CAST). Biochemical, imaging, and genetic approaches were employed to investigate the mechanistic correlations between CAST loss and mitochondrial dysfunction with aging.

*Results: Immunoblotting analysis of human and mouse livers showed that CAST expression after I/R was significantly lower in the aged group than in the young group. In aged mouse hepatocytes, 60 min of reperfusion not only reduced CAST to 5% of control levels but also induced 50% necrosis, while young cells sustained CAST and viability. Unexpectedly, the basal expression of CAST in the aged group from humans and mice was significantly greater than that in the young counterpart. Time-course analysis with cycloheximide, a translation inhibitor, implied that CAST in aged livers was intrinsically short-lived. Immunoprecipitation approach revealed that CAST in aged cells existed in a hyperacetylated form. The expression of Sirtuin1 (SIRT1), an NAD⁺-dependent deacetylase, was also significantly lower in aged cells. CAST overexpression by Ad-CAST in aged livers prevented hepatocyte death and mitochondrial permeability transition (MPT), while promoting mitophagy after I/R.

*Conclusions: Reduced SIRT1 in aged hepatocytes hyperacetylates and destabilizes CAST. Further loss of CAST after I/R instigates uncontrolled calpain activation, impaired mitophagy, MPT, and ultimately hepatocyte death. Moreover, CAST is likely a previously unidentified, new substrate of SIRT1.

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