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.

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.

Early adaptive responses in the skeletal muscle of young mice with hereditary hemochromatosis.

BACKGROUND: Hereditary hemochromatosis (HH) is characterized by iron overload that can cause multiple organ dysfunction primarily due to uncontrolled iron-mediated oxidative stress. Although HH leads to muscular weakness, disorder, and fatigue, the mechanism by which HH affects skeletal muscle physiology is largely unknown. METHODS: Using Hfe knockout mice (6-7 months old), a well-defined mouse model of HH, we examined iron status in the skeletal muscle, as well as other organs. As mitochondria are key organelle for muscular function, this study also explored how molecular markers for mitochondrial function and related systems are regulated in the HH skeletal muscle using western blots. RESULTS: Although iron overload was evident at the systemic level, only mild iron overload was observed in the skeletal muscle of HH. Of note, mitochondrial electron transport chain complex I was upregulated in the HH skeletal muscle, which was accompanied by enhanced autophagy. However, these molecular changes were not associated with oxidative stress, suggesting altered mitochondrial metabolism in the muscle in response to iron overload. CONCLUSIONS: These early adaptive responses may be important for supporting mitochondrial health before fully developing skeletal muscle dysfunction in HH. More studies are needed to determine the role of autophagy in the HH-related muscle mitochondrial dysfunction.

Protein composition of the muscle mitochondrial reticulum during postnatal development.

KEY POINTS: Muscle mitochondrial networks changed from a longitudinal, fibre parallel orientation to a perpendicular configuration during postnatal development. Mitochondrial dynamics, mitophagy and calcium uptake proteins were abundant during early postnatal development. Mitochondrial biogenesis and oxidative phosphorylation proteins were upregulated throughout muscle development. Postnatal muscle mitochondrial network formation is accompanied by a change in protein expression profile from mitochondria designed for co-ordinated cellular assembly to mitochondria highly specialized for cellular energy metabolism. ABSTRACT: Striated muscle mitochondria form connected networks capable of rapid cellular energy distribution. However, the mitochondrial reticulum is not formed at birth and the mechanisms driving network development remain unclear. In the present study, we aimed to establish the network formation timecourse and protein expression profile during postnatal development of the murine muscle mitochondrial reticulum. Two-photon microscopy was used to observe mitochondrial network orientation in tibialis anterior (TA) muscles of live mice at postnatal days (P) 1, 7, 14, 21 and 42, respectively. All muscle fibres maintained a longitudinal, fibre parallel mitochondrial network orientation early in development (P1-7). Mixed networks were most common at P14 but, by P21, almost all fibres had developed the perpendicular mitochondrial orientation observed in mature, glycolytic fibres. Tandem mass tag proteomics were then applied to examine changes in 6869 protein abundances in developing TA muscles. Mitochondrial proteins increased by 32% from P1 to P42. In addition, both nuclear- and mitochondrial-DNA encoded oxidative phosphorylation (OxPhos) components were increased during development, whereas OxPhos assembly factors decreased. Although mitochondrial dynamics and mitophagy were induced at P1-7, mitochondrial biogenesis was enhanced after P14. Moreover, calcium signalling proteins and the mitochondrial calcium uniporter had the highest expression early in postnatal development. In conclusion, mitochondrial networks transform from a fibre parallel to perpendicular orientation during the second and third weeks after birth in murine glycolytic skeletal muscle. This structural transition is accompanied by a change in protein expression profile from mitochondria designed for co-ordinated cellular assembly to mitochondria highly specialized for cellular energy metabolism.

Regulation of autophagic and mitophagic flux during chronic contractile activity-induced muscle adaptations.

Autophagy and mitophagy are important for training-inducible muscle adaptations, yet it remains unclear how these systems are regulated throughout the adaptation process. Here, we studied autophagic and mitophagic flux in the skeletal muscles of Sprague-Dawley rats (300-500 g) exposed to chronic contractile activity (CCA; 3 h/day, 9 V, 10 Hz continuous, 0.1 ms pulse duration) for 1, 2, 5, and 7 days (N = 6-8/group). In order to determine the flux rates, colchicine (COL; 0.4 mg/ml/kg) was injected 48 h before tissue collection, and we evaluated differences of autophagosomal protein abundances (LC3-II and p62) between colchicine- and saline-injected animals. We confirmed that CCA resulted in mitochondrial adaptations, including improved state 3 respiration as early as day 1 in permeabilized muscle fibers, as well significant increases in mitochondrial respiratory capacity and marker proteins in IMF mitochondria by day 7. Mitophagic and autophagic flux (LC3-II and p62) were significantly decreased in skeletal muscle following 7 days of CCA. Notably, the mitophagic system seemed to be downregulated prior (day 3-5) to changes in autophagic flux (day 7), suggesting enhanced sensitivity of mitophagy compared to autophagy with chronic muscle contraction. Although we detected no significant change in the nuclear translocation of TFEB, a regulator of lysosomal biogenesis, CCA increased total TFEB protein, as well as LAMP1, in skeletal muscle. Thus, chronic muscle activity reduces mitophagy in parallel with improved mitochondrial function, and this is supported by enhanced lysosomal degradation capacity.

Autophagy and mitophagy flux in young and aged skeletal muscle following chronic contractile activity.

KEY POINTS: A healthy mitochondrial pool is dependent on the removal of dysfunctional organelles via mitophagy, but little is known about how mitophagy is altered with ageing and chronic exercise. Chronic contractile activity (CCA) is a standardized exercise model that can elicit mitochondrial adaptations in both young and aged muscle, albeit to a lesser degree in the aged group. Assessment of mitophagy flux revealed enhanced targeting of mitochondria for degradation in aged muscle, in contrast to previous theories. Mitophagy flux was significantly reduced as an adaptation to CCA suggesting that an improvement in organelle quality reduces the need for mitochondrial turnover. CCA enhances lysosomal capacity and may ameliorate lysosomal dysfunction in aged muscle. ABSTRACT: Skeletal muscle exhibits deficits in mitochondrial quality with age. Central to the maintenance of a healthy mitochondrial pool is the removal of dysfunctional organelles via mitophagy. Little is known on how mitophagy is altered with ageing and chronic exercise. We assessed mitophagy flux using colchicine treatment in vivo following chronic contractile activity (CCA) of muscle in young and aged rats. CCA evoked mitochondrial biogenesis in young muscle, with an attenuated response in aged muscle. Mitophagy flux was higher in aged muscle and was correlated with the enhanced expression of mitophagy receptors and upstream transcriptional regulators. CCA decreased mitophagy flux in both age groups, suggesting an improvement in organelle quality. CCA also reduced the exaggerated expression of TFEB evident in aged muscle, which may be promoting the age-induced increase in lysosomal markers. Thus, aged muscle possesses an elevated drive for autophagy and mitophagy which may contribute to the decline in organelle content observed with age, but which may serve to maintain mitochondrial quality. CCA improves organelle integrity and reduces mitophagy, illustrating that chronic exercise is a modality to improve muscle quality in aged populations.

Unravelling the mechanisms regulating muscle mitochondrial biogenesis.

Skeletal muscle is a tissue with a low mitochondrial content under basal conditions, but it is responsive to acute increases in contractile activity patterns (i.e. exercise) which initiate the signalling of a compensatory response, leading to the biogenesis of mitochondria and improved organelle function. Exercise also promotes the degradation of poorly functioning mitochondria (i.e. mitophagy), thereby accelerating mitochondrial turnover, and preserving a pool of healthy organelles. In contrast, muscle disuse, as well as the aging process, are associated with reduced mitochondrial quality and quantity in muscle. This has strong negative implications for whole-body metabolic health and the preservation of muscle mass. A number of traditional, as well as novel regulatory pathways exist in muscle that control both biogenesis and mitophagy. Interestingly, although the ablation of single regulatory transcription factors within these pathways often leads to a reduction in the basal mitochondrial content of muscle, this can invariably be overcome with exercise, signifying that exercise activates a multitude of pathways which can respond to restore mitochondrial health. This knowledge, along with growing realization that pharmacological agents can also promote mitochondrial health independently of exercise, leads to an optimistic outlook in which the maintenance of mitochondrial and whole-body metabolic health can be achieved by taking advantage of the broad benefits of exercise, along with the potential specificity of drug action.

Mitochondrial iron metabolism and neurodegenerative diseases.

Iron is a key element for mitochondrial function and homeostasis, which is also crucial for maintaining the neuronal system, but too much iron promotes oxidative stress. A large body of evidence has indicated that abnormal iron accumulation in the brain is associated with various neurodegenerative diseases such as Huntington's disease, Alzheimer's disease, Parkinson's disease, and Friedreich's ataxia. However, it is still unclear how irregular iron status contributes to the development of neuronal disorders. Hence, the current review provides an update on the causal effects of iron overload in the development and progression of neurodegenerative diseases and discusses important roles of mitochondrial iron homeostasis in these disease conditions. Furthermore, this review discusses potential therapeutic targets for the treatments of iron overload-linked neurodegenerative diseases.

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'.

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.

The Functional Impact of Mitochondrial Structure Across Subcellular Scales.

Mitochondria are key determinants of cellular health. However, the functional role of mitochondria varies from cell to cell depending on the relative demands for energy distribution, metabolite biosynthesis, and/or signaling. In order to support the specific needs of different cell types, mitochondrial functional capacity can be optimized in part by modulating mitochondrial structure across several different spatial scales. Here we discuss the functional implications of altering mitochondrial structure with an emphasis on the physiological trade-offs associated with different mitochondrial configurations. Within a mitochondrion, increasing the amount of cristae in the inner membrane improves capacity for energy conversion and free radical-mediated signaling but may come at the expense of matrix space where enzymes critical for metabolite biosynthesis and signaling reside. Electrically isolating individual cristae could provide a protective mechanism to limit the spread of dysfunction within a mitochondrion but may also slow the response time to an increase in cellular energy demand. For individual mitochondria, those with relatively greater surface areas can facilitate interactions with the cytosol or other organelles but may be more costly to remove through mitophagy due to the need for larger phagophore membranes. At the network scale, a large, stable mitochondrial reticulum can provide a structural pathway for energy distribution and communication across long distances yet also enable rapid spreading of localized dysfunction. Highly dynamic mitochondrial networks allow for frequent content mixing and communication but require constant cellular remodeling to accommodate the movement of mitochondria. The formation of contact sites between mitochondria and several other organelles provides a mechanism for specialized communication and direct content transfer between organelles. However, increasing the number of contact sites between mitochondria and any given organelle reduces the mitochondrial surface area available for contact sites with other organelles as well as for metabolite exchange with cytosol. Though the precise mechanisms guiding the coordinated multi-scale mitochondrial configurations observed in different cell types have yet to be elucidated, it is clear that mitochondrial structure is tailored at every level to optimize mitochondrial function to meet specific cellular demands.

The unified myofibrillar matrix for force generation in muscle.

Human movement occurs through contraction of the basic unit of the muscle cell, the sarcomere. Sarcomeres have long been considered to be arranged end-to-end in series along the length of the muscle into tube-like myofibrils with many individual, parallel myofibrils comprising the bulk of the muscle cell volume. Here, we demonstrate that striated muscle cells form a continuous myofibrillar matrix linked together by frequently branching sarcomeres. We find that all muscle cells contain highly connected myofibrillar networks though the frequency of sarcomere branching goes down from early to late postnatal development and is higher in slow-twitch than fast-twitch mature muscles. Moreover, we show that the myofibrillar matrix is united across the entire width of the muscle cell both at birth and in mature muscle. We propose that striated muscle force is generated by a singular, mesh-like myofibrillar network rather than many individual, parallel myofibrils.

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations.

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To investigate the underlying mechanisms that bring about various muscle adaptations, a number of exercise protocols such as treadmill, wheel running, and swimming exercise have been used in the animal studies. However, these exercise models require a long period of time to achieve muscle adaptations, which may be also regulated by humoral or neurological factors, thus limiting their applications in studying the muscle-specific contraction-induced adaptations. Indirect low frequency stimulation (10 Hz) to induce chronic contractile activity (CCA) has been used as an alternative model for exercise training, as it can successfully lead to muscle mitochondrial adaptations within 7 days, independent of systemic factors. This paper details the surgical techniques required to apply the treatment of CCA to the skeletal muscle of rats, for widespread application in future studies.

Subcellular connectomic analyses of energy networks in striated muscle.

Mapping biological circuit connectivity has revolutionized our understanding of structure-function relationships. Although connectomic analyses have primarily focused on neural systems, electrical connectivity within muscle mitochondrial networks was recently demonstrated to provide a rapid mechanism for cellular energy distribution. However, tools to evaluate organelle connectivity with high spatial fidelity within single cells are currently lacking. Here, we developed a framework to quantitatively assess mitochondrial network connectivity and interactions with cellular sites of energy storage, utilization, and calcium cycling in cardiac, oxidative, and glycolytic muscle. We demonstrate that mitochondrial network configuration, individual mitochondrial size and shape, and the junctions connecting mitochondria within each network are consistent with the differing contraction demands of each muscle type. Moreover, mitochondria-lipid droplet interaction analyses suggest that individual mitochondria within networks may play specialized roles regarding energy distribution and calcium cycling within the cell and reveal the power of connectomic analyses of organelle interactions within single cells.

Effects of long-term exposures to low iron and branched-chain amino acid containing diets on aging skeletal muscle of Fisher 344 × Brown Norway rats.

Aging skeletal muscle displays an altered iron status that may promote oxidative stress and sarcopenia. A diet containing low iron (LI) could reduce muscle iron status and attenuate age-related muscle atrophy. Supplemental branched-chain amino acids (BCAA) may also alleviate sarcopenia by promoting muscle protein synthesis and iron status improvement. This study examined individual and combined effects of LI and BCAA diets on anabolic signaling and iron status in skeletal muscle of aging rats. Twenty-nine-month-old male Fisher 344 × Brown Norway rats consumed the following control-base diets: control + regular iron (35 mg iron/kg) (CR; n = 11); control + LI (∼6 mg iron/kg) (CL; n = 11); 2×BCAA + regular iron (BR; n = 10); and 2×BCAA + LI (BL; n = 12) for 12 weeks. Although LI and/or 2×BCAA did not affect plantaris muscle mass, 2×BCAA groups showed lower muscle iron content than did CR and CL groups (P < 0.05). p70 ribosomal protein S6 kinase phosphorylation was greater in 2×BCAA and LI animals compared with CR animals (P < 0.05). Interactions between IRON and BCAA were observed for proteins indicative of mitochondrial biogenesis (peroxisome proliferator-activated receptor gamma coactivator 1 alpha) and oxidative capacity (cytochrome c oxidase subunit 2 and citrate synthase) (P < 0.05) wherein the combined diet (BL) negated potential benefits of individual diets. Antioxidant capacity, superoxide dismutase activity, and oxidative injury (3-nitrotyrosine, protein carbonyls, and 4-hydroxynonenal) were similar between groups. In conclusion, 12 weeks of LI and 2×BCAA diets showed significant impacts on increasing anabolic signaling as well as ameliorating iron status; however, these interventions did not affect muscle mass.

Regulation of the autophagy system during chronic contractile activity-induced muscle adaptations.

Skeletal muscle is adaptable to exercise stimuli via the upregulation of mitochondrial biogenesis, and recent studies have suggested that autophagy also plays a role in exercise-induced muscle adaptations. However, it is still obscure how muscle regulates autophagy over the time course of training adaptations. This study examined the expression of autophagic proteins in skeletal muscle of rats exposed to chronic contractile activity (CCA; 6 h/day, 9V, 10 Hz continuous, 0.1 msec pulse duration) for 1, 3, and 7 days (n = 8/group). CCA-induced mitochondrial adaptations were observed by day 7, as shown by the increase in mitochondrial proteins (PGC-1α, COX I, and COX IV), as well as COX activity. Notably, the ratio of LC3 II/LC3 I, an indicator of autophagy, decreased by day 7 largely due to a significant increase in LC3 I. The autophagic induction marker p62 was elevated on day 3 and returned to basal levels by day 7, suggesting a time-dependent increase in autophagic flux. The lysosomal system was upregulated early, prior to changes in mitochondrial proteins, as represented by increases in lysosomal system markers LAMP1, LAMP2A, and MCOLN1 as early as by day 1, as well as TFEB, a primary regulator of lysosomal biogenesis and autophagy flux. Our findings suggest that, in response to chronic exercise, autophagy is upregulated concomitant with mitochondrial adaptations. Notably, our data reveal the surprising adaptive plasticity of the lysosome in response to chronic contractile activity which enhances muscle health by providing cells with a greater capacity for macromolecular and organelle turnover.

Impact of Aging and Exercise on Mitochondrial Quality Control in Skeletal Muscle.

Mitochondria are characterized by its pivotal roles in managing energy production, reactive oxygen species, and calcium, whose aging-related structural and functional deteriorations are observed in aging muscle. Although it is still unclear how aging alters mitochondrial quality and quantity in skeletal muscle, dysregulation of mitochondrial biogenesis and dynamic controls has been suggested as key players for that. In this paper, we summarize current understandings on how aging regulates muscle mitochondrial biogenesis, while focusing on transcriptional regulations including PGC-1α, AMPK, p53, mtDNA, and Tfam. Further, we review current findings on the muscle mitochondrial dynamic systems in aging muscle: fusion/fission, autophagy/mitophagy, and protein import. Next, we also discuss how endurance and resistance exercises impact on the mitochondrial quality controls in aging muscle, suggesting possible effective exercise strategies to improve/maintain mitochondrial health.