Impact of capillary and sarcolemmal proximity on mitochondrial structure and energetic function in skeletal muscle.

Mitochondria within skeletal muscle cells are located either between the muscle contractile apparatus (interfibrillar mitochondria, IFM) or beneath the cell membrane (subsarcolemmal mitochondria, SSM), with several structural and functional differences reported between IFM and SSM. However, recent 3D imaging studies demonstrate that mitochondria are particularly concentrated in the proximity of capillaries embedded in sarcolemmal grooves rather than in proximity to the sarcolemma itself (paravascular mitochondria, PVM). To evaluate the impact of capillary vs. sarcolemmal proximity, we compared the structure and function of skeletal muscle mitochondria located either lateral to embedded capillaries (PVM), adjacent to the sarcolemma but not in PVM pools (SSM) or interspersed between sarcomeres (IFM). Mitochondrial morphology and interactions were assessed by 3D electron microscopy coupled with machine learning segmentation, whereas mitochondrial energy conversion was assessed by two-photon microscopy of mitochondrial membrane potential, content, calcium, NADH redox and flux in live, intact cells. Structurally, although PVM and SSM were similarly larger than IFM, PVM were larger, rounder and had more physical connections to neighbouring mitochondria compared to both IFM and SSM. Functionally, PVM had similar or greater basal NADH flux compared to SSM and IFM, respectively, despite a more oxidized NADH pool and a greater membrane potential, signifying a greater activation of the electron transport chain in PVM. Together, these data indicate that proximity to capillaries has a greater impact on resting mitochondrial energy conversion and distribution in skeletal muscle than the sarcolemma alone. KEY POINTS: Capillaries have a greater impact on mitochondrial energy conversion in skeletal muscle than the sarcolemma. Paravascular mitochondria are larger, and the outer mitochondrial membrane is more connected with neighbouring mitochondria. Interfibrillar mitochondria are longer and have greater contact sites with other organelles (i.e. sarcoplasmic reticulum and lipid droplets). Paravascular mitochondria have greater activation of oxidative phosphorylation than interfibrillar mitochondria at rest, although this is not regulated by calcium.

Psat1-generated α-ketoglutarate and glutamine promote muscle stem cell activation and regeneration.

By satisfying bioenergetic demands, generating biomass, and providing metabolites serving as cofactors for chromatin modifiers, metabolism regulates adult stem cell biology. Here, we report that a branch of glycolysis, the serine biosynthesis pathway (SBP), is activated in regenerating muscle stem cells (MuSCs). Gene inactivation and metabolomics revealed that Psat1, one of the three SBP enzymes, controls MuSC activation and expansion of myogenic progenitors through production of the metabolite α-ketoglutarate (α-KG) and α-KG-generated glutamine. Psat1 ablation resulted in defective expansion of MuSCs and impaired regeneration. Psat1, α-KG, and glutamine were reduced in MuSCs of old mice. α-KG or glutamine re-established appropriate muscle regeneration of adult conditional Psat1 -/- mice and of old mice. These findings contribute insights into the metabolic role of Psat1 during muscle regeneration and suggest α-KG and glutamine as potential therapeutic interventions to ameliorate muscle regeneration during aging.

Reorganization of mitochondria-organelle interactions during postnatal development in skeletal muscle.

Skeletal muscle cellular development requires the integrated assembly of mitochondria and other organelles adjacent to the sarcomere in support of muscle contractile performance. However, it remains unclear how interactions among organelles and with the sarcomere relates to the development of muscle cell function. Here, we combine 3D volume electron microscopy, proteomic analyses, and live cell functional imaging to investigate the postnatal reorganization of mitochondria-organelle interactions in skeletal muscle. We show that while mitochondrial networks are disorganized and loosely associated with the contractile apparatus at birth, contact sites among mitochondria, lipid droplets and the sarcoplasmic reticulum are highly abundant in neonatal muscles. The maturation process is characterized by a transition to highly organized mitochondrial networks wrapped tightly around the muscle sarcomere but also to less frequent interactions with both lipid droplets and the sarcoplasmic reticulum. Concomitantly, expression of proteins involved in mitochondria-organelle membrane contact sites decreases during postnatal development in tandem with a decrease in abundance of proteins associated with sarcomere assembly despite an overall increase in contractile protein abundance. Functionally, parallel measures of mitochondrial membrane potential, NADH redox status, and NADH flux within intact cells revealed that mitochondria in adult skeletal muscle fibres maintain a more activated electron transport chain compared with neonatal muscle mitochondria. These data demonstrate a developmental redesign reflecting a shift from muscle cell assembly and frequent inter-organelle communication toward a muscle fibre with mitochondrial structure, interactions, composition and function specialized to support contractile function. KEY POINTS: Mitochondrial network organization is remodelled during skeletal muscle postnatal development. The mitochondrial outer membrane is in frequent contact with other organelles at birth and transitions to more close associations with the contractile apparatus in mature muscles. Mitochondrial energy metabolism becomes more activated during postnatal development. Understanding the developmental redesign process within skeletal muscle cells may help pinpoint specific areas of deficit in muscles with developmental disorders.

ATF4-dependent increase in mitochondrial-endoplasmic reticulum tethering following OPA1 deletion in skeletal muscle.

Mitochondria and endoplasmic reticulum (ER) contact sites (MERCs) are protein- and lipid-enriched hubs that mediate interorganellar communication by contributing to the dynamic transfer of Ca2+, lipid, and other metabolites between these organelles. Defective MERCs are associated with cellular oxidative stress, neurodegenerative disease, and cardiac and skeletal muscle pathology via mechanisms that are poorly understood. We previously demonstrated that skeletal muscle-specific knockdown (KD) of the mitochondrial fusion mediator optic atrophy 1 (OPA1) induced ER stress and correlated with an induction of Mitofusin-2, a known MERC protein. In the present study, we tested the hypothesis that Opa1 downregulation in skeletal muscle cells alters MERC formation by evaluating multiple myocyte systems, including from mice and Drosophila, and in primary myotubes. Our results revealed that OPA1 deficiency induced tighter and more frequent MERCs in concert with a greater abundance of MERC proteins involved in calcium exchange. Additionally, loss of OPA1 increased the expression of activating transcription factor 4 (ATF4), an integrated stress response (ISR) pathway effector. Reducing Atf4 expression prevented the OPA1-loss-induced tightening of MERC structures. OPA1 reduction was associated with decreased mitochondrial and sarcoplasmic reticulum, a specialized form of ER, calcium, which was reversed following ATF4 repression. These data suggest that mitochondrial stress, induced by OPA1 deficiency, regulates skeletal muscle MERC formation in an ATF4-dependent manner.

Loss of mitochondrial Ca2+ uptake protein 3 impairs skeletal muscle calcium handling and exercise capacity.

Mitochondrial calcium concentration ([Ca2+ ]m ) plays an essential role in bioenergetics, and loss of [Ca2+ ]m homeostasis can trigger diseases and cell death in numerous cell types. Ca2+ uptake into mitochondria occurs via the mitochondrial Ca2+ uniporter (MCU), which is regulated by three mitochondrial Ca2+ uptake (MICU) proteins localized in the intermembrane space, MICU1, 2, and 3. We generated a mouse model of systemic MICU3 ablation and examined its physiological role in skeletal muscle. We found that loss of MICU3 led to impaired exercise capacity. When the muscles were directly stimulated there was a decrease in time to fatigue. MICU3 ablation significantly increased the maximal force of the KO muscle and altered fibre type composition with an increase in the ratio of type IIb (low oxidative capacity) to type IIa (high oxidative capacity) fibres. Furthermore, MICU3-KO mitochondria have reduced uptake of Ca2+ and increased phosphorylation of pyruvate dehydrogenase, indicating that KO animals contain less Ca2+ in their mitochondria. Skeletal muscle from MICU3-KO mice exhibited lower net oxidation of NADH during electrically stimulated muscle contraction compared with wild-type. These data demonstrate that MICU3 plays a role in skeletal muscle physiology by setting the proper threshold for mitochondrial Ca2+ uptake, which is important for matching energy demand and supply in muscle. KEY POINTS: Mitochondrial calcium uptake is an important regulator of bioenergetics and cell death and is regulated by the mitochondrial calcium uniporter (MCU) and three calcium sensitive regulatory proteins (MICU1, 2 and 3). Loss of MICU3 leads to impaired exercise capacity and decreased time to skeletal muscle fatigue. Skeletal muscle from MICU3-KO mice exhibits a net oxidation of NADH during electrically stimulated muscle contractions, suggesting that MICU3 plays a role in skeletal muscle physiology by matching energy demand and supply.

Human Heart Failure Alters Mitochondria and Fiber 3D Structure Triggering Metabolic Shifts.

This study, utilizing SBF-SEM, reveals structural alterations in mitochondria and myofibrils in human heart failure (HF). Mitochondria in HF show changes in structure, while myofibrils exhibit increased cross-sectional area and branching. Metabolomic and lipidomic analyses indicate concomitant dysregulation in key pathways. The findings underscore the need for personalized treatments considering individualized structural changes in HF.

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.

Correction: Garza-Lopez et al. Protocols for Generating Surfaces and Measuring 3D Organelle Morphology Using Amira. Cells 2022, 11, 65.

In the original publication [...].

A Comprehensive Approach to Sample Preparation for Electron Microscopy and the Assessment of Mitochondrial Morphology in Tissue and Cultured Cells.

Mitochondria respond to metabolic demands of the cell and to incremental damage, in part, through dynamic structural changes that include fission (fragmentation), fusion (merging of distinct mitochondria), autophagic degradation (mitophagy), and biogenic interactions with the endoplasmic reticulum (ER). High resolution study of mitochondrial structural and functional relationships requires rapid preservation of specimens to reduce technical artifacts coupled with quantitative assessment of mitochondrial architecture. A practical approach for assessing mitochondrial fine structure using two dimensional and three dimensional high-resolution electron microscopy is presented, and a systematic approach to measure mitochondrial architecture, including volume, length, hyperbranching, cristae morphology, and the number and extent of interaction with the ER is described. These methods are used to assess mitochondrial architecture in cells and tissue with high energy demand, including skeletal muscle cells, mouse brain tissue, and Drosophila muscles. The accuracy of assessment is validated in cells and tissue with deletion of genes involved in mitochondrial dynamics.

Systematic Transmission Electron Microscopy-Based Identification and 3D Reconstruction of Cellular Degradation Machinery.

Various intracellular degradation organelles, including autophagosomes, lysosomes, and endosomes, work in tandem to perform autophagy, which is crucial for cellular homeostasis. Altered autophagy contributes to the pathophysiology of various diseases, including cancers and metabolic diseases. This paper aims to describe an approach to reproducibly identify and distinguish subcellular structures involved in macroautophagy. Methods are provided that help avoid common pitfalls. How to distinguish between lysosomes, lipid droplets, autolysosomes, autophagosomes, and inclusion bodies are also discussed. These methods use transmission electron microscopy (TEM), which is able to generate nanometer-scale micrographs of cellular degradation components in a fixed sample. Serial block face-scanning electron microscopy is also used to visualize the 3D morphology of degradation machinery using the Amira software. In addition to TEM and 3D reconstruction, other imaging techniques are discussed, such as immunofluorescence and immunogold labeling, which can be used to classify cellular organelles, reliably and accurately. Results show how these methods may be used to accurately quantify cellular degradation machinery under various conditions, such as treatment with the endoplasmic reticulum stressor thapsigargin or ablation of the dynamin-related protein 1.

MitoNet: A generalizable model for segmentation of individual mitochondria within electron microscopy datasets.

Volume electron microscopy provides a powerful approach to investigating physical connectivity within biological systems. In an article in this issue of Cell Systems, Conrad and Narayan overcome a major hurdle in volume electron microscopy by developing "MitoNet," a broadly applicable model for labeling individual mitochondria across volume electron microscopy datasets.

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

Sarcopenia is an age-related loss of skeletal muscle, 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 mitochondrial aging is associated with decreased mitochondrial capacity, the three-dimensional (3D) mitochondrial structure associated with morphological changes in skeletal muscle during aging still requires further elucidation. Although exercise has been the only therapy to mitigate sarcopenia, the mechanisms that govern these changes remain unclear. 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. We found that mitochondria became less complex with age. Specifically, mitochondria lost surface area, complexity, and perimeter, indicating age-related declines in ATP synthesis and interaction capacity. Concomitantly, muscle area, exercise capacity, and mitochondrial dynamic proteins showed age-related losses. Exercise stimulation restored mitofusin 2 (MFN2), which we show is required for mitochondrial structure. Furthermore, we show that this pathway is evolutionarily conserved with Marf, the MFN2 ortholog in Drosophila, as Marf 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 mitofusins.

Identification of evolutionarily conserved regulators of muscle mitochondrial network organization.

Mitochondrial networks provide coordinated energy distribution throughout muscle cells. However, pathways specifying mitochondrial networks are incompletely understood and it is unclear how they might affect contractile fiber-type. Here, we show that natural energetic demands placed on Drosophila melanogaster muscles yield native cell-types among which contractile and mitochondrial network-types are regulated differentially. Proteomic analyses of indirect flight, jump, and leg muscles, together with muscles misexpressing known fiber-type specification factor salm, identified transcription factors H15 and cut as potential mitochondrial network regulators. We demonstrate H15 operates downstream of salm regulating flight muscle contractile and mitochondrial network-type. Conversely, H15 regulates mitochondrial network configuration but not contractile type in jump and leg muscles. Further, we find that cut regulates salm expression in flight muscles and mitochondrial network configuration in leg muscles. These data indicate cell type-specific regulation of muscle mitochondrial network organization through evolutionarily conserved transcription factors cut, salm, and H15.

Mitochondrial network configuration influences sarcomere and myosin filament structure in striated muscles.

Sustained muscle contraction occurs through interactions between actin and myosin filaments within sarcomeres and requires a constant supply of adenosine triphosphate (ATP) from nearby mitochondria. However, it remains unclear how different physical configurations between sarcomeres and mitochondria alter the energetic support for contractile function. Here, we show that sarcomere cross-sectional area (CSA) varies along its length in a cell type-dependent manner where the reduction in Z-disk CSA relative to the sarcomere center is closely coordinated with mitochondrial network configuration in flies, mice, and humans. Further, we find myosin filaments near the sarcomere periphery are curved relative to interior filaments with greater curvature for filaments near mitochondria compared to sarcoplasmic reticulum. Finally, we demonstrate variable myosin filament lattice spacing between filament ends and filament centers in a cell type-dependent manner. These data suggest both sarcomere structure and myofilament interactions are influenced by the location and orientation of mitochondria within muscle cells.

Three-dimensional remodelling of the cellular energy distribution system during postnatal heart development.

The heart meets the high energy demands of constant muscle contraction and calcium cycling primarily through the conversion of fatty acids into adenosine triphosphate (ATP) by a large volume of mitochondria. As such, the spatial relationships among lipid droplets (LDs), mitochondria, the sarcotubular system and the contractile apparatus are critical to the efficient distribution of energy within the cardiomyocyte. However, the connectivity among components of the cardiac cellular energy distribution system during postnatal development remains unclear. Here, we use volume electron microscopy to demonstrate that the sarcomere branches uniting the myofibrillar network occur more than twice as frequently during early postnatal development as in mature cardiomyocytes. Moreover, we show that the mitochondrial networks arranged in parallel to the contractile apparatus are composed of larger, more compact mitochondria with greater connectivity to adjacent mitochondria in mature as compared with early postnatal cardiomyocytes. Finally, we find that connectivity among mitochondria, LDs and the sarcotubular network is greater in developing than in mature muscles. These data suggest that physical connectivity among cellular structures may facilitate the communication needed to coordinate developmental processes within the cardiac muscle cell. This article is part of the theme issue 'The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease'.

Ischemic damage to every segment of the oxidative phosphorylation cascade elevates ETC driving force and ROS production in cardiac mitochondria.

Myocardial ischemia has long-lasting negative impacts on cardiomyocyte mitochondrial ATP production. However, the location(s) of damage to the oxidative phosphorylation pathway responsible for altered mitochondrial function is unclear. Mitochondrial reactive oxygen species (ROS) production increases following ischemia, but the specific factors controlling this increase are unknown. To determine how ischemia affects the mitochondrial energy conversion cascade and ROS production, mitochondrial driving forces [redox potential and membrane potential (ΔΨ)] were measured at resting, intermediate, and maximal respiration rates in mitochondria isolated from rat hearts after 60 min of control flow (control) or no-flow ischemia (ischemia). The effective activities of the dehydrogenase enzymes, the electron transport chain (ETC), and ATP synthesis and transport were computed using the driving forces and flux. Ischemia lowered maximal mitochondrial respiration rates and diminished the responsiveness of respiration to both redox potential and ΔΨ. Ischemia decreased the activities of every component of the oxidative phosphorylation pathway: the dehydrogenase enzymes, the ETC, and ATP synthesis and transport. ROS production was linearly related to driving force down the ETC; however, ischemia mitochondria demonstrated a greater driving force down the ETC and higher ROS production. Overall, results indicate that ischemia ubiquitously damages the oxidative phosphorylation pathway, reduces mitochondrial sensitivity to driving forces, and augments the propensity for electrons to leak from the ETC. These findings underscore that strategies to improve mitochondrial function following ischemia must target the entire mitochondrial energy conversion cascade.NEW & NOTEWORTHY This integrative analysis is the first to assess how myocardial ischemia alters the mitochondrial driving forces and the degree to which individual segments of the mitochondrial energy transduction pathway contribute to diminished function following ischemia. This investigation demonstrates that increased reactive oxygen species production following ischemia is related to a lower effective activity of the electron transport chain and a greater driving force down the electron transport chain.

Regulation of the evolutionarily conserved muscle myofibrillar matrix by cell type dependent and independent mechanisms.

Skeletal muscles play a central role in human movement through forces transmitted by contraction of the sarcomere. We recently showed that mammalian sarcomeres are connected through frequent branches forming a singular, mesh-like myofibrillar matrix. However, the extent to which myofibrillar connectivity is evolutionarily conserved as well as mechanisms which regulate the specific architecture of sarcomere branching remain unclear. Here, we demonstrate the presence of a myofibrillar matrix in the tubular, but not indirect flight (IF) muscles within Drosophila melanogaster. Moreover, we find that loss of transcription factor H15 increases sarcomere branching frequency in the tubular jump muscles, and we show that sarcomere branching can be turned on in IF muscles by salm-mediated conversion to tubular muscles. Finally, we demonstrate that neurochondrin misexpression results in myofibrillar connectivity in IF muscles without conversion to tubular muscles. These data indicate an evolutionarily conserved myofibrillar matrix regulated by both cell-type dependent and independent mechanisms.

Subcellular Specialization of Mitochondrial Form and Function in Skeletal Muscle Cells.

Across different cell types and within single cells, mitochondria are heterogeneous in form and function. In skeletal muscle cells, morphologically and functionally distinct subpopulations of mitochondria have been identified, but the mechanisms by which the subcellular specialization of mitochondria contributes to energy homeostasis in working muscles remains unclear. Here, we discuss the current data regarding mitochondrial heterogeneity in skeletal muscle cells and highlight potential new lines of inquiry that have emerged due to advancements in cellular imaging technologies.

A Universal Approach to Analyzing Transmission Electron Microscopy with ImageJ.

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.