A Workshop to Enrich Physiological Understanding Through Hands-On Learning About Mitochondria-Endoplasmic Reticulum Contact Sites.

Physiology is an important field for students to gain a better understanding of biological mechanisms. Yet, many students often find it difficult to learn from lectures, resulting in poor retention. Here, we focus on the utilization of a learning workshop model to teach students at different levels ranging from middle school to undergraduate. We specifically designed a workshop to teach students about mitochondria endoplasmic reticulum contact (MERC) sites. The workshop was implemented for middle-school students in a laboratory setting that incorporated a pre-test to gauge prior knowledge, instructional time, hands-on activities, interactive learning from experts, and a post-test. We observed that the students remained engaged during the session relied on interactive methods, teamed with their peers to complete tasks, and delighted in the experience. Implications for the design of future physiological workshops are further offered.

Ablation of Sam50 is associated with fragmentation and alterations in metabolism in murine and human myotubes.

The sorting and assembly machinery (SAM) Complex is responsible for assembling ß-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 in Sam50-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 and Sam50-deficient myotubes. The results revealed increased mitochondrial fragmentation and autophagosome formation in Sam50-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 in Sam50-deficient myotubes. Furthermore, impairment of oxidative capacity was observed upon Sam50 ablation 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 of Sam50-deficiency, this study enhances our understanding of mitochondrial function in skeletal muscle.

Considerations for developing mitochondrial transplantation techniques for individualized medicine.

Tweetable abstract Mitochondrial transplantation has been used to treat various diseases associated with mitochondrial dysfunction. Here, we highlight the considerations in quality control mechanisms that should be considered in the context of mitochondrial transplantation.

A workshop to showcase the diversity of scientists to middle school students.

Identity matters in science, technology, engineering, mathematics, and medicine (STEMM) because it can affect an individual's long-term sense of belonging, which may in turn affect their persistence in STEMM. Early K-12 science classes often teach students about the foundational discoveries of the field, which have been predominately made, or at least published, by White men. This homogeneity can leave underrepresented individuals in STEMM feeling isolated, and underrepresented K-12 students may feel as though they cannot enter STEMM fields. This study aimed to examine these feelings of inclusivity in STEMM through an interactive workshop that asked middle schoolers to identify scientists from images of individuals with various racial and gender identities. We found that a plurality of students had a positive experience discussing diversity in science and recognizing underrepresented individuals as scientists.NEW & NOTEWORTHY We observed positive sentiments from middle school students following a workshop that showcased diversity in science. This workshop uniquely encourages students to recognize that physiologists and scientists today are much more diverse than textbooks typically demonstrate and can be adapted for middle schoolers, high schoolers, and college students.

3D Mitochondrial Structure in Aging Human Skeletal Muscle: Insights into MFN-2 Mediated Changes.

Age-related atrophy of skeletal muscle, is characterized by loss of mass, strength, endurance, and oxidative capacity during aging. Notably, bioenergetics and protein turnover studies have shown that mitochondria mediate this decline in function. Although exercise has been the only therapy to mitigate sarcopenia, the mechanisms that govern how exercise serves to promote healthy muscle aging are unclear. Mitochondrial aging is associated with decreased mitochondrial capacity, so we sought to investigate how aging affects mitochondrial structure and potential age-related regulators. Specifically, the three-dimensional (3D) mitochondrial structure associated with morphological changes in skeletal muscle during aging requires further elucidation. We hypothesized that aging causes structural remodeling of mitochondrial 3D architecture representative of dysfunction, and this effect is mitigated by exercise. We used serial block-face scanning electron microscopy to image human skeletal tissue samples, followed by manual contour tracing using Amira software for 3D reconstruction and subsequent analysis of mitochondria. We then applied a rigorous in vitro and in vivo exercise regimen during aging. Across 5 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 we 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 in Drosophila, 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.

Defining Mitochondrial Cristae Morphology Changes Induced by Aging in Brown Adipose Tissue.

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.

Is space the final frontier for mitochondrial study?

Tweetable abstract This perspective considers several avenues for future research on mitochondrial dynamics, stress, and DNA in outer space.

A pilot study on our non-traditional, varied writing accountability group for historically excluded and underrepresented persons in STEMM.

Underrepresented faculty have higher burnout rates and lower grant attainment rates when compared with their non-minority counterparts. Many in science, technology, engineering, mathematics, and medicine (STEMM) disciplines, including underrepresented individuals, often have difficulty dedicating time to the writing process, with trainees often being relegated to laboratory tasks in their training years, resulting in a lack of practice in academic writing. Notably, past studies have shown that grant attainment rates of underrepresented individuals are lower than their majority counterparts. Here, we sought to consider a mechanism targeted to underrepresented individuals, although applicable to everyone, to help overcome traditional barriers to writing in STEMM. The authors have hosted a writing accountability group (WAG) that uniquely provides a format focused on physical activity and different forms of writing to strengthen both career development and award/funding attainment. Our objectives were to evaluate this unique format, thus creating a resource for individuals and institutions to learn about WAGs and expand upon the framework to formulate their own WAG. To do this, we performed a small pilot study (n = 21) to investigate attitudes towards the WAG. We present the results of a survey conducted among underrepresented WAG participants, which spanned different career stages and was highly diverse demographically. Our results show that following attendance of our WAG, individuals did not note a significant change in scales pertaining to John Henryism (high-effort coping), resilience, sense of belonging, or grit. However, significant increases were noted in the self-perceived ability to handle stress, confidence in applying for awards, appreciation for mentoring, and satisfaction of WAGs. Taken together, the results of this study suggest that our unique WAG format can have some positive results as a career and writing development opportunity and may be able to support underrepresented individuals in attaining funding at higher education institutions.

3D reconstruction of murine mitochondria reveals changes in structure during aging linked to the MICOS complex.

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.

Diversity, Equity and Inclusion in the Laboratory: Strategies to Enhance Inclusive Laboratory Culture.

Building a diverse laboratory that is equitable is critical for the retention of talent and the growth of trainees professionally and personally. Here, we outline several strategies including enhancing understanding of cultural competency and humility, establishing laboratory values, and developing equitable laboratory structures to create an inclusive laboratory environment to enable trainees to achieve their highest success.

Protocol for isolating mice skeletal muscle myoblasts and myotubes via differential antibody validation.

Isolation of skeletal muscles allows for the exploration of many complex diseases. Here, we present a protocol for isolating mice skeletal muscle myoblasts and myotubes that have been differentiated through antibody validation. We describe steps for collecting and preparing murine skeletal tissue, myoblast cell maintenance, plating, and cell differentiation. We then detail procedures for cell incubation, immunostaining, slide preparation and storage, and imaging for immunofluorescence validation.

Project Strengthen: An STEMM-focused career development workshop to prepare underrepresented minority students for graduate school.

Maximizing Access to Research Careers (MARC) programs are aimed to increase diversity in science, technology, engineering, math, and medicine (STEMM) fields. However, limited programs and eligibility requirements limit the students who may apply to similar programs. At Winston-Salem State University, we piloted a series of workshops, collectively termed Project Strengthen, to emulate some of the key aspects of MARC programs. Following the workshop, Project Strengthen students showed a significant increase in their understanding of essential educational development skills, such as writing personal statements, applying to graduate school, studying for the GRE, and seeking summer internships. This suggests Project Strengthen may be a potential lower cost comparable option than MARC to make up for current deficiencies in preparedness for graduate school. We also provide educational materials from Project Strengthen, including a clear framework for this seminar series, six ready-made PowerPoints to share with trainees that have been demonstrated to be effective.

A workshop on mitochondria for students to improve understanding of science and hypothesis forming.

There remains a clear deficiency in recruiting middle school students in science, technology, engineering, mathematics, and medicine fields, especially for those students entering physiology from underrepresented backgrounds. A large part of this may be arising from a disconnect between how science is typically practiced at a collegiate and K-12 level. Here, we have envisioned mitochondria and their diverse subcellular structures as an involver for middle school students. We present the framework for a workshop that familiarizes students with mitochondria, employing three-dimensional visual-spatial learning and real-time critical thinking and hypothesis forming. This workshop had the goal of familiarizing middle school students with the unique challenges the field currently faces and better understanding the actuality of being a scientist through critical analysis including hypothesis forming. Findings show that middle school students responded positively to the program and felt as though they had a better understanding of mitochondria. Future implications for hands-on programs to involve underrepresented students in science are discussed, as well as potential considerations to adapt it for high school and undergraduate students.NEW & NOTEWORTHY Here we employ a workshop that utilizes blended and tactile learning to teach middle schoolers about mitochondrial structure. By creating an approachable and fun workshop that can be utilized for middle school students, we seek to encourage them to join a career in physiology.

Defining Mitochondrial Cristae Morphology Changes Induced by Aging in Brown Adipose Tissue.

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, we hypothesized that significant morphological changes in BAT mitochondria and cristae would be present with aging. We developed a quantitative three-dimensional (3D) electron microscopy approach to map cristae network organization in mouse BAT to test this hypothesis. Using this methodology, we investigated the 3D morphology of mitochondrial cristae 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, we found increases 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.

Three-dimensional mitochondria reconstructions of murine cardiac muscle changes in size across aging.

With sparse treatment options, cardiac disease remains a significant cause of death among humans. As a person ages, mitochondria breakdown and the heart becomes less efficient. Heart failure is linked to many mitochondria-associated processes, including endoplasmic reticulum stress, mitochondrial bioenergetics, insulin signaling, autophagy, and oxidative stress. The roles of key mitochondrial complexes that dictate the ultrastructure, such as the mitochondrial contact site and cristae organizing system (MICOS), in aging cardiac muscle are poorly understood. To better understand the cause of age-related alteration in mitochondrial structure in cardiac muscle, we used transmission electron microscopy (TEM) and serial block facing-scanning electron microscopy (SBF-SEM) to quantitatively analyze the three-dimensional (3-D) networks in cardiac muscle samples of male mice at aging intervals of 3 mo, 1 yr, and 2 yr. Here, we present the loss of cristae morphology, the inner folds of the mitochondria, across age. In conjunction with this, the three-dimensional (3-D) volume of mitochondria decreased. These findings mimicked observed phenotypes in murine cardiac fibroblasts with CRISPR/Cas9 knockout of Mitofilin, Chchd3, Chchd6 (some members of the MICOS complex), and Opa1, which showed poorer oxidative consumption rate and mitochondria with decreased mitochondrial length and volume. In combination, these data show the need to explore if loss of the MICOS complex in the heart may be involved in age-associated mitochondrial and cristae structural changes.NEW & NOTEWORTHY 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.

Ablation of Sam50 is associated with fragmentation and alterations in metabolism in murine and human myotubes.

The Sorting and Assembly Machinery (SAM) Complex is responsible for assembling ß-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 (MICOS) 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 (SBF-SEM) and computer-assisted 3D renderings were employed to compare mitochondrial structure and networking in Sam50-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 and Sam50-deficient myotubes. The results revealed increased mitochondrial fragmentation and autophagosome formation in Sam50-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 in Sam50-deficient myotubes. Furthermore, impairment of oxidative capacity was observed upon Sam50 ablation 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 of Sam50-deficiency, this study enhances our understanding of mitochondrial function in skeletal muscle.

Components of Isolated Skeletal Muscle Differentiated Through Antibody Validation.

Isolation of skeletal muscles allows for the exploration of many complex diseases. Fibroblasts and myoblast play important roles in skeletal muscle morphology and function. However, skeletal muscles are complex and made up of many cellular populations and validation of these populations is highly important. Therefore, in this article, we discuss a comprehensive method to isolate mice skeletal muscle, create satellite cells for tissue culture, and use immunofluorescence to validate our approach.

Creating Optimal Conditions for OPA1 Isoforms by Western Blot in Skeletal Muscle Cells and Tissue.

OPA1 is a dynamin-related GTPase that modulates various mitochondrial functions and is involved in mitochondrial morphology. There are eight different isoforms of OPA1 in humans and five different isoforms in mice that are expressed as short or long-form isoforms. These isoforms contribute to OPA1's ability to control mitochondrial functions. However, isolating OPA1 all long and short isoforms through western blot has been a difficult task. To address this issue, we outline an optimized western blot protocol to isolate 5 different isoforms of OPA1 on the basis of different antibodies. This protocol can be used to study changes in mitochondrial structure and function.

Optimizing In Situ Proximity Ligation Assays for Mitochondria, ER, or MERC Markers in Skeletal Muscle Tissue and Cells.

Proximity ligation assays (PLA) use specific antibodies to detect endogenous protein-protein interactions. PLA is a highly useful biochemical technique that allows two proteins within close proximity to be visualized with fluorescent probes amplified by PCR. While this technique has gained prominence, the use of PLA in mouse skeletal muscle (SkM) is novel. In this article, we discuss how the PLA method can be used in SkM to study the protein-protein interactions within mitochondria-endoplasmic reticulum contact sites (MERCs).

What does Juneteenth mean in STEMM?

Juneteenth commemorates the freeing of the last large group of enslaved people in 1865 at the end of the American Civil War. We asked several Black scientists what Juneteenth means to them in the context of science, technology, engineering, mathematics, and medicine (STEMM)? Their answers run the emotional gamut.