Abstract 342: Increased Mitochondrial Permeability Transition Pore Opening Dominates Ischemia-Reperfusion Injury in the Aged Female Rat Heart

Abstract

Background: Cardiovascular disease remains the leading cause of death in older post-menopausal women. Ischemia/Reperfusion (I/R) injury triggers mitochondrial calcium (Ca 2+ ) overload inducing mitochondrial permeability transition pore (MPTP) opening, mitochondrial dysfunction, and cell death potentially by necrosis, apoptosis, and/or autophagy. Purpose: We sought to determine if age-associated estrogen deficiency increases mitochondrial Ca 2+ sensitivity, providing a possible mechanism for increased vulnerability to I/R injury in older women. Methods: Mitochondrial respiration (MR) was assessed in isolated mitochondria from ventricles of adult (6 mo; n=15) and aged (24 mo; n=18) ovary-intact or ovariectomized (OVX) female F344 rats. MR at complexes I and II was compared in the absence (State 2) and presence (State 3) of ADP to calculate respiratory control index (RCI; state3/state 2). Reduced RCI following Ca 2+ addition was used to assess Ca 2+ sensitivity, while mitochondrial Ca 2+ retention capacity was measured to quantify MPTP opening (CRC; n=4-5/group) prior to and following coronary artery ligation (55 min I and 6 hr R). Apoptosis was examined using DNA laddering and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Markers of autophagy were evaluated by western blotting and mitochondrial morphology through electron microscopy (EM). Results: Significant age-dependent decreases in RCI for complex I (12%) and complex II (8%) were observed in the absence of Ca 2+ , and correlated with increased necrosis in aged hearts revealed by triphenyltetrazolium chloride (TTC) staining (p < 0.05). Ca 2+ exposure decreased MR (18-30%; p < 0.05) in Complex I of aged and OVX mitochondria vs adults. Furthermore, CRC worsened with age requiring less Ca 2+ to open the MPTP. Reduced DNA laddering and TUNEL staining combined with increased beclin-1 and cathepsinD expression in aged vs. adult further support a dominant role for necrosis over apoptosis underlying cell death in aged females (n=4-5/group). EM revealed morphological alterations with age and OVX. Conclusion: Decreased MR and increased MPTP opening with aging are likely causal in necrotic cell death mechanisms associated with I/R injury observed in post-menopausal women.