Mitochondria play a central role in age-related pathologies, loss of resilience, and the decline in quality of life in
older adults. As we age changes in mitochondrial function lead to disruption of redox and energy homeostasis,
altered metabolite levels, impaired calcium regulation, and increased sensitivity to permeability transition, all of
which contribute to tissue dysfunction. Mitochondria are dynamic organelles that continuously adapt to changing
cellular demands by altering protein assembly and interactions to modify their function. Despite the obvious
importance little is known about how age-related changes in mitochondrial protein interactions underlie changes
in function. To address this fundamental question we propose to apply a state of the art novel quantitative
chemical cross-linking with mass spectrometry approach (XL-MS) to quantify the changes in the mitochondrial
interactome in heart and skeletal muscles with age. By combining this innovative XL-MS approach with targeted
functional assays and interventions developed over the last several years to manipulate mitochondria in vivo and
in vitro we are uniquely positioned to identify the changes in mitochondrial protein interactions that underlie age-
related mitochondrial dysfunction. Our preliminary data indicate specific disruption of multiple protein interaction
networks involved in ADP transport, ATP synthesis, glutamate metabolism, and complexes of the electron
transport system with age. Many of these protein complexes that XL-MS indicates are disrupted in aging directly
interact with the mitochondrial targeted peptide SS-31 that we have shown reverses mitochondrial dysfunction
in heart and skeletal muscle. Our overall hypothesis is that changes in the mitochondrial interactome driven by
elevated mitochondrial redox stress underlie decreased ATP production and altered metabolite levels in
mitochondria from aged heart and skeletal muscle. In aim 1 we will apply XL-MS and site-specific assays to
measure the link between the interactome and function in mitochondria from aged mouse heart and skeletal
muscle. As a specific test of new mechanistic insights available from this approach we will use gene-edited iPSC
derived cardiomyocytes to test the causal relationship between altered interactions in the glutamate
dehydrogenase regulatory region, the largest change identified in the aged mitochondrial interactome, and
impaired glutamate and a-ketoglutarate metabolism. In aim 2 we will use mitochondrial targeted interventions to
reverse mitochondrial dysfunction, SS-31 and AAV-mitochondrial targeted catalase, and induce mitochondrial
redox stress to identify the most functionally important changes in the mitochondrial interactome from aim 1. In
aim 3 we use human muscle biopsies to test whether changes in the mitochondrial interactome identified in aims
1 and 2 translate into aged human skeletal muscle. We will separate older adults into low and high performing
groups to compare the mitochondrial interactomes and function with those of young adults. Results from these
experiments will have a transformative impact on the field by providing the first window into the links between
the mitochondrial protein interaction network and age-related mitochondrial dysfunction.
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