Mitochondrial structural plasticity is a critical adaptive response to cellular stress, yet the transcriptional networks governing the formation of specialized mitochondrial architectures remain poorly defined. Here, we identified and demonstrated that activating transcription factor 4 (ATF4), the master regulator of the integrated stress response, directly regulates mitochondrial morphological remodeling through a novel ATF4-NRF1/Nrf2-MFN2 signaling axis. Using serial block-face scanning electron microscopy and three-dimensional reconstruction in Drosophila flight muscle, primary myotubes, and human skeletal muscle, we show that overexpression of ATF4 promotes significant mitochondrial elongation, increased cristae concentration, enhanced mitochondrial-endoplasmic reticulum contact site (MERC) formation, and the initiation of Mitochondrial Nanotunnels. In contrast, loss of ATF4 results in mitochondrial fragmentation and impaired aerobic capacity. Chromatin immunoprecipitation sequencing reveals direct ATF4 binding at the promoters of the genes encoding NRF1 and Nrf2, which in turn regulate MFN2 expression. Small-molecule inhibition studies further establish that activation of this hierarchical pathway is both necessary and sufficient for stress-induced mitochondrial structural adaptation. Together, these findings position ATF4 as a master regulator of mitochondrial architectural plasticity, providing a direct mechanistic link between cellular stress signaling and organelle remodeling.

Mutations in the small heat shock protein α-crystallin B (CryAB) result in cataracts, cardiomyopathies, and myofibrillar myopathies (MFMs), all of which are marked by protein aggregation. To investigate pathological mechanisms, we expressed four human CryAB disease alleles in Drosophila skeletal muscle. All variants resulted in the accumulation of protein aggregates. Mutations within the conserved α-crystallin domain (ACD) caused CryAB-positive structures that colocalized with an amyloidogenic form of human Desmin. The amyloid-like nature of these CryAB variants was further supported by thioflavin T spectroscopy and Congo red staining, the latter of which was also evident in other MFM-causing genes in zebrafish muscles and human biopsies. Muscle-enriched CryAB amyloid-like structures co-localized with extracellular vesicle (EV) markers and were detected in the hemolymph, suggesting an EV-mediated export mechanism. This is the first report of CryAB amyloid formation in skeletal muscle and broadens amyloid dynamics beyond the nervous system.

Abstract Transmembrane protein 65 (TMEM65) depletion in a patient caused severe mitochondrial encephalomyopathy, highlighting its clinical importance. Recent studies show TMEM65 acts as a mitochondrial Na+/Ca2+ exchanger in vitro. Here, we generated conditional Tmem65 knockout mice to define its role in neuromuscular tissues in vivo. Both whole-body and nervous system–specific Tmem65 knockouts exhibited severe growth retardation and seizure-associated sudden death at ~3 weeks, establishing TMEM65 as indispensable for neuronal function. Additionally, skeletal muscle–specific knockout produced adult-onset myopathy preceded by elevated mitochondrial Ca2+. Consistently, TMEM65 ablation caused loss of Na+-dependent mitochondrial Ca2+ export. Notably, blocking mitochondrial Ca2+ entry by mitochondrial calcium uniporter (MCU) knockout rescued the early lethality of whole-body Tmem65 ablation, extending lifespan from ~3 weeks to >1 year. These data reveal an essential physiological role for TMEM65 and suggest that modulating mitochondrial Ca2+ may offer therapeutic value for TMEM65 misexpression and other mitochondrial diseases associated with Ca2+ overload.

Mitochondrial network morphology is widely used as an indicator of cellular health; however, quantifying mitochondrial architecture within intact tissues remains technically challenging. Terminal Schwann cells (tSCs), which are essential for the maintenance and regeneration of neuromuscular junctions, are particularly difficult to analyze in situ due to their anatomical location and sensitivity to tissue disruption. This protocol describes a reproducible approach for labeling and quantifying three-dimensional mitochondrial network morphology in whole-mount skeletal muscle and in tSCs in mice using standard confocal microscopy. The method employs in vivo delivery of a membrane potential-sensitive mitochondrial dye followed by rapid tissue processing and high-resolution confocal imaging. Image stacks are analyzed to quantify mitochondrial network connectivity, area, and fragmentation. The protocol is first validated in dystrophic and healthy skeletal muscle to confirm expected differences in mitochondrial morphology and is subsequently adapted to visualize and quantify mitochondrial networks in tSCs identified using S100β reporter mice. This approach enables the analysis of mitochondrial morphology within intact neuromuscular tissues without requiring transgenic mitochondrial reporters or specialized imaging platforms. The protocol requires only equipment commonly available in university core facilities and can be adapted to other thin or superficially accessible tissues.

Cellular organelles are not just static structures; they are highly dynamic and directly linked to cellular functions. Changes in their morphology can be early indicators of diseases. Recent advancements in light microscopy techniques have transformed organelle research from qualitative descriptions to precise, quantitative measurements, enabling nanoscale resolution, high-throughput image analysis, and live-cell compatibility. This enables accurate measurement of organelle morphology, dynamics, and spatial organization using modern imaging and analysis techniques. By quantifying organelles, we go beyond simply visualizing to measuring and statistically comparing cellular features across different samples. This article addresses a wide range of cellular organelles across all major experimental systems, specifically mentioning mitochondria, myofibers, actin filaments, endoplasmic reticulum, and Golgi apparatus, by integrating experimental design, optimized sample preparation, high-resolution imaging, and validated Fiji/ImageJ-based analysis workflows. For each organelle, step-by-step protocols specify reagents, equipment, acquisition parameters, and expected results. Although recent advances, such as expansion microscopy, correlative light-electron microscopy, and AI-powered segmentation, offer gains in throughput and resolution, this workflow demonstrates that Fiji-based analysis remains fully capable of delivering high-precision organelle quantification. The entire workflow can be completed within 2-4 weeks, from initial design through validation and the production of measurements suitable for cross-study comparisons. Overall, these protocols establish a flexible approach to standardizing organelle quantification so as to understand multiple organelles simultaneously in their cellular contexts. © 2026 Wiley Periodicals LLC. Basic Protocol 1: Mitochondrial quantification Basic Protocol 2: Lipid droplet identification and image processing Basic Protocol 3: Myofibril quantification Basic Protocol 4: Golgi apparatus morphometry Basic Protocol 5: Endoplasmic reticulum network analysis Alternate Protocol: Super-resolution imaging protocol.

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 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.

Mitochondrial dysfunction is well described in many chronic illnesses including musculoskeletal, neurodegenerative, and cardiovascular diseases. Mitochondrial network morphology has been implicated as a biomarker of disease, correlating increased mitochondrial fragmentation to impaired cellular function. While advancements in imaging techniques further our understanding of mitochondrial dynamics in live cells, easily accessible approaches for accurate quantification of in situ mitochondrial networks in low abundance tissues are lacking. The purpose of this study was to validate a proof-of-concept method capable of quantifying 3D mitochondrial network morphology in whole mount skeletal muscle and then applying it to mitochondrial morphology analysis in cell types otherwise difficult to image within their native environment, terminal Schwann cells (tSCs). Herein, we report that mitochondrial networks were fragmented in dystrophic mouse muscle compared to healthy controls, as observed by others, and correlated with muscle pathology as expected. Using S100β reporter mice to identify Schwann cells, we labeled tSC mitochondrial networks in vivo prior to rapid imaging in situ with high-resolution confocal microscopy. Moreover, these methods offer a comprehensive and novel approach enabling the quantification of mitochondria network morphology across multiple cell types (like muscle fibers and tSCs) using standard microscopy available in university core facilities. Summary Local injections of mitochondrial dye are used to label terminal Schwann cells for confocal microscopy imaging after proof of concept was demonstrated in skeletal muscle tissue from mice with healthy or diseased muscle. ### Competing Interest Statement The authors have declared no competing interest. Sydney and J.L. Huffines Institute for Sports Medicine and Human Performance at Texas A and M University College of Education and Human Development at Texas A and M University

Introduction and Objective: Patients with type 2 diabetes (T2D) exhibit altered skeletal muscle mitochondrial dynamics which worsens insulin action and depletes mitochondrial content. Mitochondrial Rho GTPase (MIRO1) regulates organelle trafficking, but its role in maintaining skeletal muscle glucoregulatory functions is unknown. This study examined the relationship between MIRO1 expression and insulin sensitivity in T2D. Methods: In a cross-sectional study, participants were grouped as (1) healthy weight (BMI: 22.4 ± 1.7), (2) obesity without T2D (BMI: 31.9 ± 3.5), or (3) T2D (HbA1c: 7.3 ± 1.3%; 1.3 ± 1.2 glucose-lowering medications). Body composition, aerobic capacity, and insulin sensitivity were assessed during a 3-day inpatient evaluation. Fasting muscle biopsies assessed mitochondrial ultrastructure and bioenergetics. In a randomized controlled trial, a separate cohort of patients with obesity and T2D underwent a 12-week aerobic exercise program (ExT) or standard therapy (CTRL). Outcomes included MIRO1 expression, mitochondrial ultrastructure, and bioenergetics. Results: Glucose disposal rates inversely correlated with T2D status and MIRO1 expression. ExT increased VO2peak, glucose disposal, and reduced MIRO1 expression relative to CTRL (p<). ExT also increased N-linked OXPHOS (p<0.05), N-linked conductance (p<), and reduced mitochondrial fission and lipid droplet accumulation (p<0.001) compared to CTRL. Conclusion: Skeletal muscle MIRO1 expression inversely correlates with insulin sensitivity in T2D. Exercise training reversed this accompanied by improvements in glucose disposal, VO2peak, bioenergetic function, and mitochondrial dynamics. A.L. Taylor: None. E.C. Heintz: None. W.S. Dantas: None. E.R. Zunica: None. J.T. Mey: None. B. Vandanmagsar: None. H.A. Parry: None. K. Belmont: None. R.A. Beyl: None. B. Glancy: None. C.L. Axelrod: None. J.P. Kirwan: None.

Imbalanced skeletal muscle mitochondrial dynamics contributed to the onset and progression of type 2 diabetes (T2D) by mechanisms that remained incompletely understood. Here, we examined the role of mitochondrial Rho GTPase 1 (Miro1), an outer mitochondrial membrane enzyme, in the regulation of skeletal muscle insulin action and glucose homeostasis in T2D. Miro1 accumulated in the skeletal muscle of mice and humans with obesity and T2D, a phenomenon driven by impaired insulin-mediated interaction between AKT and Miro1 at the outer mitochondrial membrane. To determine whether Miro1 accumulation was reversible and functionally linked to metabolic improvements, we prospectively evaluated the impact of exercise training on skeletal muscle Miro1 expression, mitochondrial function, and insulin sensitivity in patients with T2D. Patients with T2D (N=24) were randomized to 12 weeks of standard care or exercise training. At baseline and after 12 weeks, we assessed changes in whole-body metabolic and mitochondrial function. Exercise training reduced skeletal muscle Miro1 accumulation (64.3% vs. -53.2% change from baseline; p=0.001) and enhanced mitochondrial oxidative capacity (-37.7% vs. 216.3% change from baseline; p=0.005) and insulin sensitivity (-18.5% vs. 80.0% change from baseline; p=0.007). To further establish a causal role for Miro1 in glucose homeostasis, we generated muscle-specific Miro1 loss- of-function models in mice and cells. Muscle-specific deletion of Miro1 improved insulin action and oxidative capacity in both models. Taken together, these findings supported a key regulatory role for skeletal muscle Miro1 in the pathophysiology of T2D.

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.Psat1ablation 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 conditionalPsat1−/−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.

Across normal aging, striated muscles undergo structural remodeling associated with loss of force production. However, it is unknown how the organization of contractile myofibrillar networks, linked to their efficiency, is modified during aging. Using serial block-face scanning electron microscopy (SBF-SEM), we assess myofibril size, shape, and connectivity across different muscle types in young and geriatric mice and humans. Regardless of skeletal muscle fiber type in mice, age was associated with increased myofibrillar connectivity, with 24 months of age, as compared to 3 months, displaying more sarcomere branches. Distinctive age-related trends in myofibril size and shape were observed among each muscle type. Notably, there was a decrease in myofibril circularity from 3 months of age to 24 months of age in the gastrocnemius muscles of mice, contrasting with an increase in circularity in the soleus muscles during the same time frame. Additionally, while the soleus myofibrils in an aged cohort had a higher cross-sectional area, a reduction was observed in the gastrocnemius muscles. Cardiac muscles displayed no changes in sarcomere connectivity from 3 months to 24 months, although myofibril circularity and cross-sectional area were increased during this time. In human vastus lateralis muscles, sarcomere branching was positively correlated with advanced age. However, there were no consistent changes in myofibril size or shape across a wide age range from 16 to 68 years old. Overall, these data suggest that aging is associated with increased connectivity of the contractile networks within mammalian skeletal muscle. ### Competing Interest Statement The authors have declared no competing interest.

Transmembrane protein 65 (TMEM65) depletion in a patient carrying a homozygous variant in the Tmem65 splice site resulted in severe mitochondrial encephalomyopathy, indicating the clinical importance of TMEM65. However, the function of TMEM65 remains unknown. Here, we generated a TMEM65 reporter mouse as well as whole-body and tissue-specific Tmem65 knockout (KO) mice to investigate the localization and function of TMEM65. We show that TMEM65 is localized to mitochondria in heart, skeletal muscle, and throughout the brain. Both whole-body and nervous system-specific Tmem65 KO result in severe growth retardation and sudden death following seizures ~3 weeks after birth, indicating TMEM65 is indispensable for normal brain function. In addition, we find that skeletal muscle-specific Tmem65 KO leads to progressive, adult-onset myopathy preceded by elevated mitochondrial calcium levels despite unaltered expression of known mitochondrial or cellular calcium handling proteins. Consistently, we demonstrate that ablation of TMEM65 results in a loss of sodium-dependent mitochondrial calcium export. Finally, we show that blocking mitochondrial calcium entry through removal of the mitochondrial calcium uniporter (MCU) rescues the early lethality of whole-body TMEM65 ablation. Our data not only reveal the essential role of TMEM65 in mammalian physiology, but also suggest modulating mitochondrial calcium may offer a potential therapeutical approach to address defects associated with TMEM65 misexpression.

Cardiac troponin I (cTnI) is a sarcomeric protein critical to myocyte contraction. Unexpectedly, we found that some cTnI localized to the mitochondrial matrix in the heart, inhibited mitochondrial functions when stably expressed in non-cardiac cells and increased opening of the mitochondrial permeability transition pore under oxidative stress. Direct, specific, and saturable binding of cTnI to ATP synthase was demonstrated in vitro , using immune-captured ATP synthase, and in cells using proximity ligation assay. cTnI binding doubled F 1 F 0 ATPase activity, whereas skeletal troponin I and several human mutant cTnI variants associated with familial hypertrophic cardiomyopathy did not. A rationally-designed ten amino acid peptide, P888, inhibited cTnI binding to ATP synthase, inhibited cTnI-induced increase in ATPase activity in vitro , and reduced cardiac injury following transient ischemia in vivo . We therefore suggest that mitochondria-associated cTnI may inhibit cardiac ATP synthase under basal conditions; pharmacological agents that release this inactivating effect of cTnI and thus preventing ATP hydrolysis during cardiac ischemia may increase the reservoir of functional mitochondria to reduce cardiac injury. Significance Statement Cardiac troponin I (cTnI) is a key sarcomeric protein involved in the regulation of myocardial contractility. We found that some cTnI is present in the mitochondrial matrix where it binds to ATP synthase, disrupting mitochondrial function; inhibition of the cTnI-ATP synthase interaction with a selective peptide inhibitor reduces cardiac dysfunction following ischemia and reperfusion injury. Several pathogenic cTnI mutations associated with hypertrophic cardiomyopathy do not affect ATP synthase activity, suggesting a potential mechanism that contributes to the diverse pathologies associated with these mutations.

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.

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.

Introduction: Chronic activation of DRP1 promotes mitochondrial network fragmentation and restricts skeletal muscle glucose uptake in patients with type 2 diabetes (T2D). The purpose of this study was to determine if exercise reverses hyperactivation of skeletal muscle mitochondrial fission in patients with T2D. Methods: 24 sedentary adults (49 ± 10 years) with obesity (BMI 36 ± 6 kg/m2) and T2D (HbA1C 7.3 ± 1.3%; 1.3 ± 1.2 glucose lowering medications) were randomized to 12 wks of exercise training (Ex; n=11) or standard care (Ctrl; n=13). Participants randomized to Ex performed supervised aerobic exercise 5 d/wk for 60 min/session at 80-85% HRMAX. Participants underwent a 3-day inpatient stay consisting of DXA, VO2peak, and 2-stage hyperinsulinemic-euglycemic clamp at baseline and post intervention. Skeletal muscle biopsies were obtained for Western blot, high-resolution respirometry, and ion beam scanning electron microscopy with machine learning segmentation. Relative changes from baseline were calculated, and comparisons were made with unpaired Student’s t-tests. The primary outcome was change in pDRP1Ser616 expression. Results: Age, sex, body weight, and diabetes status were similar at baseline (p>0.05). Ex increased VO2peak, lean mass, and peripheral insulin sensitivity and reduced fat mass relative to Ctrl (all p<0.05). Skeletal muscle pDRP1Ser616 was reduced in Ex (p<0.05) independent of PGC-1α and AMPK. Ex increased maximal NADH-linked OXPHOS (p<0.05) and improved succinate- and complex III-linked OXPHOS (p<0.1). Mitochondria were elongated and had reduced sphericity after Ex (p<0.001). Conclusions: Exercise training reversed hyperactivation of skeletal muscle mitochondrial fission and improved respiratory capacity independent of biogenesis. Collectively, these data indicate that exercise improves skeletal muscle insulin sensitivity in T2D, in part, by restoring mitochondrial dynamics and networking. Disclosure E.C. Heintz: None. W.S. Dantas: None. E.R.M. Zunica: None. K. Belmont: None. J.T. Mey: None. R.A. Beyl: None. D.S. Hsia: None. H.A. Parry: None. B. Glancy: None. C.L. Hoppel: Advisory Panel; Cytokinetics Inc. C.L. Axelrod: None. J.P. Kirwan: None. Funding National Institutes of Health (DK108089 and GM104940)