Lam, Jacob

Abstract During aging, muscle gradually undergoes sarcopenia, the loss of function associated with loss of mass, strength, endurance, and oxidative capacity. However, the 3D structural alterations of mitochondria associated with aging in skeletal muscle and cardiac tissues are not well described. Although mitochondrial aging is associated with decreased mitochondrial capacity, the genes responsible for the morphological changes in mitochondria during aging are poorly characterized. We measured changes in mitochondrial morphology in aged murine gastrocnemius, soleus, and cardiac tissues using serial block‐face scanning electron microscopy and 3D reconstructions. We also used reverse transcriptase‐quantitative PCR, transmission electron microscopy quantification, Seahorse analysis, and metabolomics and lipidomics to measure changes in mitochondrial morphology and function after loss of mitochondria contact site and cristae organizing system (MICOS) complex genes, Chchd3 , Chchd6 , and Mitofilin . We identified significant changes in mitochondrial size in aged murine gastrocnemius, soleus, and cardiac tissues. We found that both age‐related loss of the MICOS complex and knockouts of MICOS genes in mice altered mitochondrial morphology. Given the critical role of mitochondria in maintaining cellular metabolism, we characterized the metabolomes and lipidomes of young and aged mouse tissues, which showed profound alterations consistent with changes in membrane integrity, supporting our observations of age‐related changes in muscle tissues. We found a relationship between changes in the MICOS complex and aging. Thus, it is important to understand the mechanisms that underlie the tissue‐dependent 3D mitochondrial phenotypic changes that occur in aging and the evolutionary conservation of these mechanisms between Drosophila and mammals.

This article shows how mitochondria in murine cardiac changes, importantly elucidating age-related changes. It also is the first to show that the MICOS complex may play a role in outer membrane mitochondrial structure.

High-resolution 3D images of organelles are of paramount importance in cellular biology. Although light microscopy and transmission electron microscopy (TEM) have provided the standard for imaging cellular structures, they cannot provide 3D images. However, recent technological advances such as serial block-face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM) provide the tools to create 3D images for the ultrastructural analysis of organelles. Here, we describe a standardized protocol using the visualization software, Amira, to quantify organelle morphologies in 3D, thereby providing accurate and reproducible measurements of these cellular substructures. We demonstrate applications of SBF-SEM and Amira to quantify mitochondria and endoplasmic reticulum (ER) structures.

Transmission electron microscopy (TEM) is widely used as an imaging modality to provide high-resolution details of subcellular components within cells and tissues. Mitochondria and endoplasmic reticulum (ER) are organelles of particular interest to those investigating metabolic disorders. A straightforward method for quantifying and characterizing particular aspects of these organelles would be a useful tool. In this protocol, we outline how to accurately assess the morphology of these important subcellular structures using open source software ImageJ, originally developed by the National Institutes of Health (NIH). Specifically, we detail how to obtain mitochondrial length, width, area, and circularity, in addition to assessing cristae morphology and measuring mito/endoplasmic reticulum (ER) interactions. These procedures provide useful tools for quantifying and characterizing key features of sub-cellular morphology, leading to accurate and reproducible measurements and visualizations of mitochondria and ER.