Neonatology Fellow University of Rochester Medical Center University of Rochester Medical Center Rochester, New York, United States
Background: Mitochondria play an integral role in the regulation of cardiac development at the cellular level. At birth, mitochondrial signaling pathways initiate the final stage of myocyte differentiation to increase cardiac output to meet the increased metabolic demands. These changes in metabolism are regulated by the chaperone protein cyclophilin D (CypD). Inhibition or deletion of CypD accelerates assembly of the electron transport chain (ETC) contributing to the developmentally regulated activation of the oxidative phosphorylation in the heart.
Objective: This study seeks to establish a developmental timeline of ETC assembly in the neonatal heart.
Design/Methods: Hearts were harvested for mitochondrial isolation from wild type and CypD homozygous knockout (CypD KO) mice ranging from the fetal period to the adult. Assays of enzymatic activity of each complex within the ETC were quantified using spectrophotometry and normalized to total protein. To measure physical assembly of complexes, high resolution clear native polyacrylamide gel electrophoresis (HCRN PAGE) followed by in-gel assays or immunoblotting were utilized.
Results: Homogenates and isolated mitochondria were obtained from mouse hearts from fetal (embryonic day (E) 16.5), neonatal (postnatal days (P) 1 and 7), and older (weanling and adult) ages. Using enzyme assays with heart homogenates from wild type mice, we found an increase in ETC complex I activity (NADH-ubiquinone oxidoreductase) as gestational age increased from E16.5 to adult (P <0.01, number of samples for each age >5). In contrast, complex II activity was stable across the gestational ages, with no significant difference between the ages. Using isolated mitochondria, we found greater ETC complex I activity in CypD KO mice compared to age matched WT mice (P <0.05, number of samples for each age 5). HCRN PAGE followed by in-gel ETC complex assays or immunoblotting demonstrated increasing assembly of complex V into dimers and tetramers with increasing age. Conclusion(s): It is important to understand the changes that occur in mitochondrial function, as mitochondria regulate the final stage of development that occurs after birth. This study provides insight that with increasing gestational age the assembly and activity of the electron transport chain in cardiac myocytes increases to meet the metabolic demands for survival of the neonate. As these changes are regulated by CypD, our future studies will further define changes in ETC assembly and activity during this time period and how altering CypD modulates this process.
Authors/Institutions: Jonathan R. Burris, University of Rochester Medical Center, Rochester, New York, United States; George A. Porter, University of Rochester Medical Center, Rochester, New York, United States