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Abstract OPA1 is a dynamin‐related GTPase that modulates mitochondrial dynamics and cristae integrity. Humans carry eight different isoforms of OPA1 and mice carry five, all of which are expressed as short‐ or long‐form isoforms. These isoforms contribute to OPA1's ability to control mitochondrial energetics and DNA maintenance. However, western blot isolation of all long and short isoforms of OPA1 can be difficult. To address this issue, we developed an optimized western blot protocol based on improving running time to isolate five different isoforms of OPA1 in mouse cells and tissues. This protocol can be applied to study changes in mitochondrial structure and function. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Western Blot Protocol for Isolating OPA1 Isoforms in Mouse Primary Skeletal Muscle Cells 

Abstract While some established undergraduate summer programs are effective across many institutions, these programs may only be available to some principal investigators or may not fully address the diverse needs of incoming undergraduates. This article outlines a 10‐week science, technology, engineering, mathematics, and medicine (STEMM) education program designed to prepare undergraduate students for graduate school through a unique model incorporating mentoring dyads and triads, cultural exchanges, and diverse activities while emphasizing critical thinking, research skills, and cultural sensitivity. Specifically, we offer a straightforward and adaptable guide that we have used for mentoring undergraduate students in a laboratory focused on mitochondria and microscopy, but which may be customized for other disciplines. Key components include self‐guided projects, journal clubs, various weekly activities such as mindfulness training and laboratory techniques, and a focus on individual and cultural expression. Beyond this unique format, this 10‐week program also seeks to offer an intensive research program that emulates graduate‐level experiences, offering an immersive environment for personal and professional development, which has led to numerous achievements for past students, including publications and award‐winning posters. 

Serial block face scanning electron microscopy (SBF‐SEM), also referred to as serial block‐face electron microscopy, is an advanced ultrastructural imaging technique that enables three‐dimensional visualization that provides larger x- and y‐axis ranges than other volumetric EM techniques. While SEM is first introduced in the 1930s, SBF‐SEM is developed as a novel method to resolve the 3D architecture of neuronal networks across large volumes with nanometer resolution by Denk and Horstmann in 2004. Here, the authors provide an accessible overview of the advantages and challenges associated with SBF‐SEM. Beyond this, the applications of SBF‐SEM in biochemical domains as well as potential future clinical applications are briefly reviewed. Finally, the alternative forms of artificial intelligence‐based segmentation which may contribute to devising a feasible workflow involving SBF‐SEM, are also considered. 

ABSTRACT Age‐related skeletal muscle atrophy, known as sarcopenia, is characterized by loss of muscle mass, strength, endurance, and oxidative capacity. Although exercise has been shown to mitigate sarcopenia, the underlying governing mechanisms are poorly understood. Mitochondrial dysfunction is implicated in aging and sarcopenia; however, few studies explore how mitochondrial structure contributes to this dysfunction. In this study, we sought to understand how aging impacts mitochondrial three‐dimensional (3D) structure and its regulators in skeletal muscle. We hypothesized that aging leads to remodeling of mitochondrial 3D architecture permissive to dysfunction and is ameliorated by exercise. Using serial block‐face scanning electron microscopy (SBF‐SEM) and Amira software, mitochondrial 3D reconstructions from patient biopsies were generated and analyzed. Across five human cohorts, we correlate differences in magnetic resonance imaging, mitochondria 3D structure, exercise parameters, and plasma immune markers between young (under 50 years) and old (over 50 years) individuals. We found that mitochondria are less spherical and more complex, indicating age‐related declines in contact site capacity. Additionally, aged samples showed a larger volume phenotype in both female and male humans, indicating potential mitochondrial swelling. Concomitantly, muscle area, exercise capacity, and mitochondrial dynamic proteins showed age‐related losses. Exercise stimulation restored mitofusin 2 (MFN2), one such of these mitochondrial dynamic proteins, which we show is required for the integrity of mitochondrial structure. Furthermore, we show that this pathway is evolutionarily conserved, as Marf, the MFN2 ortholog inDrosophila, knockdown alters mitochondrial morphology and leads to the downregulation of genes regulating mitochondrial processes. Our results define age‐related structural changes in mitochondria and further suggest that exercise may mitigate age‐related structural decline through modulation of mitofusin 2. 

Abstract The physical characteristics of brown adipose tissue (BAT) are defined by the presence of multilocular lipid droplets (LDs) within the brown adipocytes and a high abundance of iron‐containing mitochondria, which give it its characteristic color. Normal mitochondrial function is, in part, regulated by organelle‐to‐organelle contacts. For example, the contact sites that mediate mitochondria–LD interactions are thought to have various physiological roles, such as the synthesis and metabolism of lipids. Aging is associated with mitochondrial dysfunction, and previous studies show that there are changes in mitochondrial structure and the proteins that modulate organelle contact sites. However, how mitochondria–LD interactions change with aging has yet to be fully clarified. Therefore, we sought to define age‐related changes in LD morphology and mitochondria–lipid interactions in BAT. We examined the three‐dimensional morphology of mitochondria and LDs in young (3‐month) and aged (2‐year) murine BAT using serial block face‐scanning electron microscopy and the Amira program for segmentation, analysis, and quantification. Our analyses showed reductions in LD volume, area, and perimeter in aged samples in comparison to young samples. Additionally, we observed changes in LD appearance and type in aged samples compared to young samples. Notably, we found differences in mitochondrial interactions with LDs, which could implicate that these contacts may be important for energetics in aging. Upon further investigation, we also found changes in mitochondrial and cristae structure for the mitochondria interacting with LDs. Overall, these data define the nature of LD morphology and organelle–organelle contacts during aging and provide insight into LD contact site changes that interconnect biogerontology with mitochondrial function, metabolism, and bioactivity in aged BAT. 

Abstract The sorting and assembly machinery (SAM) Complex is responsible for assembling β‐barrel proteins in the mitochondrial membrane. Comprising three subunits, Sam35, Sam37, and Sam50, the SAM complex connects the inner and outer mitochondrial membranes by interacting with the mitochondrial contact site and cristae organizing system complex. Sam50, in particular, stabilizes the mitochondrial intermembrane space bridging (MIB) complex, which is crucial for protein transport, respiratory chain complex assembly, and regulation of cristae integrity. While the role of Sam50 in mitochondrial structure and metabolism in skeletal muscle remains unclear, this study aims to investigate its impact. Serial block‐face‐scanning electron microscopy and computer‐assisted 3D renderings were employed to compare mitochondrial structure and networking inSam50‐deficient myotubes from mice and humans with wild‐type (WT) myotubes. Furthermore, autophagosome 3D structure was assessed in human myotubes. Mitochondrial metabolic phenotypes were assessed using Gas Chromatography‐Mass Spectrometry‐based metabolomics to explore differential changes in WT andSam50‐deficient myotubes. The results revealed increased mitochondrial fragmentation and autophagosome formation inSam50‐deficient myotubes compared to controls. Metabolomic analysis indicated elevated metabolism of propanoate and several amino acids, including ß‐Alanine, phenylalanine, and tyrosine, along with increased amino acid and fatty acid metabolism inSam50‐deficient myotubes. Furthermore, impairment of oxidative capacity was observed uponSam50ablation in both murine and human myotubes, as measured with the XF24 Seahorse Analyzer. Collectively, these findings support the critical role of Sam50 in establishing and maintaining mitochondrial integrity, cristae structure, and mitochondrial metabolism. By elucidating the impact ofSam50‐deficiency, this study enhances our understanding of mitochondrial function in skeletal muscle. 

Abstract Alzheimer’s Disease (AD) is a global health issue, affecting over 6 million in the United States, with that number expected to increase as the aging population grows. As a neurodegenerative disorder that affects memory and cognitive functions, it is well established that AD is associated with cardiovascular risk factors beyond only cerebral decline. However, the study of cerebrovascular techniques for AD is still evolving. Here, we provide reproducible methods to measure impedance-based pulse wave velocity (PWV), a marker of arterial stiffness, in the systemic vascular (aortic PWV) and in the cerebral vascular (cerebral PWV) systems. Using aortic impedance and this relatively novel technique of cerebral impedance to comprehensively describe the systemic vascular and the cerebral vascular systems, we examined the sex-dependent differences in 5x transgenic mice (5XFAD) with AD under normal and high-fat diet, and in wild-type mice under a normal diet. Additionally, we validated our method for measuring cerebrovascular impedance in a model of induced stress in 5XFAD. Together, our results show that sex and diet differences in wildtype and 5XFAD mice account for very minimal differences in cerebral impedance. Interestingly, 5XFAD, and not wildtype, male mice on a chow diet show higher cerebral impedance, suggesting pathological differences. Opposingly, when we subjected 5XFAD mice to stress, we found that females showed elevated cerebral impedance. Using this validated method of measuring impedance-based aortic and cerebral PWV, future research may explore the effects of modifying factors including age, chronic diet, and acute stress, which may mediate cardiovascular risk in AD. New and NoteworthyHere, we presented a new technique which is an application of the concept of aortic impedance to determining cerebral impedance. While aortic PWV is typically utilized to study aortic stiffness, we also developed a technique of cerebral PWV to study cerebral vascular stiffness. This method may be useful in improving the rigor of studies that seek to have a dual focus on cardiovascular and cerebral function. 

Abstract Mitochondria are required for energy production and even give brown adipose tissue (BAT) its characteristic color due to their high iron content and abundance. The physiological function and bioenergetic capacity of mitochondria are connected to the structure, folding, and organization of its inner‐membrane cristae. During the aging process, mitochondrial dysfunction is observed, and the regulatory balance of mitochondrial dynamics is often disrupted, leading to increased mitochondrial fragmentation in aging cells. Therefore, it is hypothesized that significant morphological changes in BAT mitochondria and cristae will be present with aging. A quantitative 3D electron microscopy approach is developed to map cristae network organization in mouse BAT to test this hypothesis. Using this methodology, the 3D morphology of mitochondrial cristae is investigated in adult (3‐month) and aged (2‐year) murine BAT tissue via serial block face‐scanning electron microscopy (SBF‐SEM) and 3D reconstruction software for manual segmentation, analysis, and quantification. Upon investigation, an increase is found in mitochondrial volume, surface area, and complexity and decreased sphericity in aged BAT, alongside significant decreases in cristae volume, area, perimeter, and score. Overall, these data define the nature of the mitochondrial structure in murine BAT across aging.