Need for speed: Human fast-twitch mitochondria favor power over efficiency.

OBJECTIVE: Human skeletal muscle consists of a mixture of slow- and fast-twitch fibers with distinct capacities for contraction mechanics, fermentation, and oxidative phosphorylation. While the divergence in mitochondrial volume favoring slow-twitch fibers is well established, data on the fiber type-specific intrinsic mitochondrial function and morphology are highly limited with existing data mainly being generated in animal models. This highlights the need for more human data on the topic. METHODS: Here, we utilized THRIFTY, a rapid fiber type identification protocol to detect, sort, and pool fast- and slow-twitch fibers within 6 h of muscle biopsy sampling. Respiration of permeabilized fast- and slow-twitch fiber pools was then analyzed with high-resolution respirometry. Using standardized western blot procedures, muscle fiber pools were subsequently analyzed for control proteins and key proteins related to respiratory capacity. RESULTS: Maximal complex I+II respiration was 25% higher in human slow-twitch fibers compared to fast-twitch fibers. However, per mitochondrial volume, the respiratory rate of mitochondria in fast-twitch fibers was approximately 50% higher for complex I+II, which was primarily mediated through elevated complex II respiration. Furthermore, the abundance of complex II protein and proteins regulating cristae structure were disproportionally elevated in mitochondria of the fast-twitch fibers. The difference in intrinsic respiratory rate was not reflected in fatty acid-or complex I respiration. CONCLUSION: Mitochondria of human fast-twitch muscle fibers compensate for their lack of volume by substantially elevating intrinsic respiratory rate through increased reliance on complex II.

Azilsartan prevents muscle loss and fast- to slow-twitch muscle fiber shift in natural ageing sarcopenic rats.

Sarcopenia is a musculoskeletal disease that reduces muscle mass and strength in older individuals. The study investigates the effects of azilsartan (AZL) on skeletal muscle loss in natural sarcopenic rats. Male Sprague-Dawley rats aged 4-6 months and 18-21 months were selected as young-matched control and natural-aged (sarcopenic) rats, respectively. Rats were allocated into young and old control (YC and OC) and young and old AZL treatment (YT and OT) groups, which received vehicles and AZL (8 mg/kg, orally) for 6 weeks. Rats were then sacrificed after muscle function analysis. Serum and gastrocnemius (GN) muscles were isolated for further endpoints. AZL significantly improved muscle grip strength and antioxidant levels in sarcopenic rats. AZL also restored the levels of insulin, testosterone, and muscle biomarkers such as myostatin and creatinine kinase in sarcopenic rats. Furthermore, AZL treatment improved the cellular and ultrastructure of GN muscle and prevented the shift of type II (glycolytic) myofibers to type I (oxidative) myofibers. The results showed that AZL intervention restored protein synthesis in natural sarcopenic rats by increasing p-Akt-1 and decreasing muscle RING-finger protein-1 and tumor necrosis factor alpha immunoexpressions. In conclusion, the present findings showed that AZL could be an effective intervention in treating age-related muscle impairments.

Inherited myogenic abilities in muscle precursor cells defined by the mitochondrial complex I-encoding protein.

Skeletal muscle comprises different muscle fibers, including slow- and fast-type muscles, and satellite cells (SCs), which exist in individual muscle fibers and possess different myogenic properties. Previously, we reported that myoblasts (MBs) from slow-type enriched soleus (SOL) had a high potential to self-renew compared with cells derived from fast-type enriched tibialis anterior (TA). However, whether the functionality of myogenic cells in adult muscles is attributed to the muscle fiber in which they reside and whether the characteristics of myogenic cells derived from slow- and fast-type fibers can be distinguished at the genetic level remain unknown. Global gene expression analysis revealed that the myogenic potential of MBs was independent of the muscle fiber type they reside in but dependent on the region of muscles they are derived from. Thus, in this study, proteomic analysis was conducted to clarify the molecular differences between MBs derived from TA and SOL. NADH dehydrogenase (ubiquinone) iron-sulfur protein 8 (Ndufs8), a subunit of NADH dehydrogenase in mitochondrial complex I, significantly increased in SOL-derived MBs compared with that in TA-derived cells. Moreover, the expression level of Ndufs8 in MBs significantly decreased with age. Gain- and loss-of-function experiments revealed that Ndufs8 expression in MBs promoted differentiation, self-renewal, and apoptosis resistance. In particular, Ndufs8 suppression in MBs increased p53 acetylation, followed by a decline in NAD/NADH ratio. Nicotinamide mononucleotide treatment, which restores the intracellular NAD+ level, could decrease p53 acetylation and increase myogenic cell self-renewal ability in vivo. These results suggested that the functional differences in MBs derived from SOL and TA governed by the mitochondrial complex I-encoding gene reflect the magnitude of the decline in SC number observed with aging, indicating that the replenishment of NAD+ is a possible approach for improving impaired cellular functions caused by aging or diseases.

Effects of acute cigarette smoke concentrate exposure on mitochondrial energy transfer in fast- and slow-twitch skeletal muscle.

The mechanisms underlying cigarette smoke-induced mitochondrial dysfunction in skeletal muscle are still poorly understood. Accordingly, this study aimed to examine the effects of cigarette smoke on mitochondrial energy transfer in permeabilized muscle fibers from skeletal muscles with differing metabolic characteristics. The electron transport chain (ETC) capacity, ADP transport, and respiratory control by ADP were assessed in fast- and slow-twitch muscle fibers from C57BL/6 mice (n = 11) acutely exposed to cigarette smoke concentrate (CSC) using high-resolution respirometry. CSC decreased complex I-driven respiration in the white gastrocnemius (CONTROL:45.4 ± 11.2 pmolO2.s-1.mg-1 and CSC:27.5 ± 12.0 pmolO2.s-1.mg-1; p = 0.01) and soleus (CONTROL:63.0 ± 23.8 pmolO2.s-1.mg-1 and CSC:44.6 ± 11.1 pmolO2.s-1.mg-1; p = 0.04). In contrast, the effect of CSC on Complex II-linked respiration increased its relative contribution to muscle respiratory capacity in the white gastrocnemius muscle. The maximal respiratory activity of the ETC was significantly inhibited by CSC in both muscles. Furthermore, the respiration rate dependent on the ADP/ATP transport across the mitochondrial membrane was significantly impaired by CSC in the white gastrocnemius (CONTROL:-70 ± 18 %; CSC:-28 ± 10 %; p < 0.001), but not the soleus (CONTROL:47 ± 16 %; CSC:31 ± 7 %; p = 0.08). CSC also significantly impaired mitochondrial thermodynamic coupling in both muscles. Our findings underscore that acute CSC exposure directly inhibits oxidative phosphorylation in permeabilized muscle fibers. This effect was mediated by significant perturbations of the electron transfer in the respiratory complexes, especially at complex I, in both fast and slow twitch muscles. In contrast, CSC-induced inhibition of the exchange of ADP/ATP across the mitochondrial membrane was fiber-type specific, with a large effect on fast-twitch muscles.

Large Maf transcription factor family is a major regulator of fast type IIb myofiber determination.

Myofibers are broadly characterized as fatigue-resistant slow-twitch (type I) fibers and rapidly fatiguing fast-twitch (type IIa/IIx/IIb) fibers. However, the molecular regulation of myofiber type is not entirely understood; particularly, information on regulators of fast-twitch muscle is scarce. Here, we demonstrate that the large Maf transcription factor family dictates fast type IIb myofiber specification in mice. Remarkably, the ablation of three large Mafs leads to the drastic loss of type IIb myofibers, resulting in enhanced endurance capacity and the reduction of muscle force. Conversely, the overexpression of each large Maf in the type I soleus muscle induces type IIb myofibers. Mechanistically, a large Maf directly binds to the Maf recognition element on the promoter of myosin heavy chain 4, which encodes the type IIb myosin heavy chain, driving its expression. This work identifies the large Maf transcription factor family as a major regulator for fast type IIb muscle determination.

Bridging the muscle genome to phenome across multiple biological scales.

Muscle is highly hierarchically organized, with functions shaped by genetically controlled expression of protein ensembles with different isoform profiles at the sarcomere scale. However, it remains unclear how isoform profiles shape whole-muscle performance. We compared two mouse hindlimb muscles, the slow, relatively parallel-fibered soleus and the faster, more pennate-fibered tibialis anterior (TA), across scales: from gene regulation, isoform expression and translation speed, to force-length-velocity-power for intact muscles. Expression of myosin heavy-chain (MHC) isoforms directly corresponded with contraction velocity. The fast-twitch TA with fast MHC isoforms had faster unloaded velocities (actin sliding velocity, Vactin; peak fiber velocity, Vmax) than the slow-twitch soleus. For the soleus, Vactin was biased towards Vactin for purely slow MHC I, despite this muscle's even fast and slow MHC isoform composition. Our multi-scale results clearly identified a consistent and significant dampening in fiber shortening velocities for both muscles, underscoring an indirect correlation between Vactin and fiber Vmax that may be influenced by differences in fiber architecture, along with internal loading due to both passive and active effects. These influences correlate with the increased peak force and power in the slightly more pennate TA, leading to a broader length range of near-optimal force production. Conversely, a greater force-velocity curvature in the near-parallel fibered soleus highlights the fine-tuning by molecular-scale influences including myosin heavy and light chain expression along with whole-muscle characteristics. Our results demonstrate that the individual gene, protein and whole-fiber characteristics do not directly reflect overall muscle performance but that intricate fine-tuning across scales shapes specialized muscle function.

Exploring the super-relaxed state of myosin in myofibrils from fast-twitch, slow-twitch, and cardiac muscle.

Muscle myosin heads, in the absence of actin, have been shown to exist in two states, the relaxed (turnover ∼0.05 s-1) and super-relaxed states (SRX, 0.005 s-1) using a simple fluorescent ATP chase assay (Hooijman, P. et al (2011) Biophys. J.100, 1969-1976). Studies have normally used purified proteins, myosin filaments, or muscle fibers. Here we use muscle myofibrils, which retain most of the ancillary proteins and 3-D architecture of muscle and can be used with rapid mixing methods. Recording timescales from 0.1 to 1000 s provides a precise measure of the two populations of myosin heads present in relaxed myofibrils. We demonstrate that the population of SRX states is formed from rigor cross bridges within 0.2 s of relaxing with fluorescently labeled ATP, and the population of SRX states is relatively constant over the temperature range of 5 °C-30 °C. The SRX population is enhanced in the presence of mavacamten and reduced in the presence of deoxy-ATP. Compared with myofibrils from fast-twitch muscle, slow-twitch muscle, and cardiac muscles, myofibrils require a tenfold lower concentration of mavacamten to be effective, and mavacamten induced a larger increase in the population of the SRX state. Mavacamten is less effective, however, at stabilizing the SRX state at physiological temperatures than at 5 °C. These assays require small quantities of myofibrils, making them suitable for studies of model organism muscles, human biopsies, or human-derived iPSCs.

Differential effect of canagliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, on slow and fast skeletal muscles from nondiabetic mice.

There has been a concern that sodium-glucose cotransporter 2 (SGLT2) inhibitors could reduce skeletal muscle mass and function. Here, we examine the effect of canagliflozin (CANA), an SGLT2 inhibitor, on slow and fast muscles from nondiabetic C57BL/6J mice. In this study, mice were fed with or without CANA under ad libitum feeding, and then evaluated for metabolic valuables as well as slow and fast muscle mass and function. We also examined the effect of CANA on gene expressions and metabolites in slow and fast muscles. During SGLT2 inhibition, fast muscle function is increased, as accompanied by increased food intake, whereas slow muscle function is unaffected, although slow and fast muscle mass is maintained. When the amount of food in CANA-treated mice is adjusted to that in vehicle-treated mice, fast muscle mass and function are reduced, but slow muscle was unaffected during SGLT2 inhibition. In metabolome analysis, glycolytic metabolites and ATP are increased in fast muscle, whereas glycolytic metabolites are reduced but ATP is maintained in slow muscle during SGLT2 inhibition. Amino acids and free fatty acids are increased in slow muscle, but unchanged in fast muscle during SGLT2 inhibition. The metabolic effects on slow and fast muscles are exaggerated when food intake is restricted. This study demonstrates the differential effects of an SGLT2 inhibitor on slow and fast muscles independent of impaired glucose metabolism, thereby providing new insights into how they should be used in patients with diabetes, who are at a high risk of sarcopenia.

Comprehensive Simulation of Ca2+ Transients in the Continuum of Mouse Skeletal Muscle Fiber Types.

Mag-Fluo-4 has revealed differences in the kinetics of the Ca2+ transients of mammalian fiber types (I, IIA, IIX, and IIB). We simulated the changes in [Ca2+] through the sarcomere of these four fiber types, considering classical (troponin -Tn-, parvalbumin -Pv-, adenosine triphosphate -ATP-, sarcoplasmic reticulum Ca2+ pump -SERCA-, and dye) and new (mitochondria -MITO-, Na+/Ca2+ exchanger -NCX-, and store-operated calcium entry -SOCE-) Ca2+ binding sites, during single and tetanic stimulation. We found that during a single twitch, the sarcoplasmic peak [Ca2+] for fibers type IIB and IIX was around 16 µM, and for fibers type I and IIA reached 10-13 µM. The release rate in fibers type I, IIA, IIX, and IIB was 64.8, 153.6, 238.8, and 244.5 µM ms-1, respectively. Both the pattern of change and the peak concentrations of the Ca2+-bound species in the sarcoplasm (Tn, PV, ATP, and dye), the sarcolemma (NCX, SOCE), and the SR (SERCA) showed the order IIB ≥ IIX > IIA > I. The capacity of the NCX was 2.5, 1.3, 0.9, and 0.8% of the capacity of SERCA, for fibers type I, IIA, IIX, and IIB, respectively. MITO peak [Ca2+] ranged from 0.93 to 0.23 µM, in fibers type I and IIB, respectively, while intermediate values were obtained in fibers IIA and IIX. The latter numbers doubled during tetanic stimulation. In conclusion, we presented a comprehensive mathematical model of the excitation-contraction coupling that integrated most classical and novel Ca2+ handling mechanisms, overcoming the limitations of the fast- vs. slow-fibers dichotomy and the use of slow dyes.

Naringin induces skeletal muscle fiber type transformation via AMPK/PGC-1α signaling pathway in mice and C2C12 myotubes.

A large number of studies have shown that polyphenols can regulate skeletal muscle fiber type transformation through AMPK signal. However, the effects and mechanism of naringin (a natural polyphenol) on muscle fiber type transformation still remains unclear. Thus, we hypothesized that naringin would induce the transformation of skeletal muscle fibers from type II to type I by AMPK signaling. C2C12 myotubes and BALB/c mice models were used to test this hypothesis. We found that naringin significantly increased the protein expression of slow myosin heavy chain (MyHC), myoglobin and troponin I type I slow skeletal (Troponin I-SS) and the activities of succinate dehydrogenase (SDH) and malate dehydrogenase (MDH), and significantly decreased fast MyHC protein expression and lactate dehydrogenase (LDH) activity, accompanied by the activation of AMPK and the activity of peroxisome proliferator activated receptor-γ coactivator-1α (PGC-1α) in mice and C2C12 myotubes. Further inhibition of AMPK activity by compound C showed that the above effects were significantly inhibited in C2C12 myotubes. In conclusion, naringin promotes the transformation of skeletal muscle fibers from type II to type I through AMPK/PGC-1α signaling pathway, which not only enriches the nutritional and physiological functions of naringin, but also provides a theoretical basis for the regulation of muscle fiber type transformation by nutritional approaches.

Fast skeletal myosin-binding protein-C regulates fast skeletal muscle contraction.

Fast skeletal myosin-binding protein-C (fMyBP-C) is one of three MyBP-C paralogs and is predominantly expressed in fast skeletal muscle. Mutations in the gene that encodes fMyBP-C, MYBPC2, are associated with distal arthrogryposis, while loss of fMyBP-C protein is associated with diseased muscle. However, the functional and structural roles of fMyBP-C in skeletal muscle remain unclear. To address this gap, we generated a homozygous fMyBP-C knockout mouse (C2-/-) and characterized it both in vivo and in vitro compared to wild-type mice. Ablation of fMyBP-C was benign in terms of muscle weight, fiber type, cross-sectional area, and sarcomere ultrastructure. However, grip strength and plantar flexor muscle strength were significantly decreased in C2-/- mice. Peak isometric tetanic force and isotonic speed of contraction were significantly reduced in isolated extensor digitorum longus (EDL) from C2-/- mice. Small-angle X-ray diffraction of C2-/- EDL muscle showed significantly increased equatorial intensity ratio during contraction, indicating a greater shift of myosin heads toward actin, while MLL4 layer line intensity was decreased at rest, indicating less ordered myosin heads. Interfilament lattice spacing increased significantly in C2-/- EDL muscle. Consistent with these findings, we observed a significant reduction of steady-state isometric force during Ca2+-activation, decreased myofilament calcium sensitivity, and sinusoidal stiffness in skinned EDL muscle fibers from C2-/- mice. Finally, C2-/- muscles displayed disruption of inflammatory and regenerative pathways, along with increased muscle damage upon mechanical overload. Together, our data suggest that fMyBP-C is essential for maximal speed and force of contraction, sarcomere integrity, and calcium sensitivity in fast-twitch muscle.

Chronic electrical stimulation reduces reliance on anaerobic metabolism in locust jumping muscle.

Chronic electrical stimulation (CES) is a well-documented method for changing mammalian muscle from more fast-twitch to slow-twitch metabolic and contractile profiles. Although both mammalian and insect muscles have many similar anatomical and physiological properties, it is unknown if CES produces similar muscle plasticity changes in insects. To test this idea, we separated Schistocerca americana grasshoppers into two groups (n = 37 to 47): one that was subjected to CES for 180 min each day for five consecutive days and one group that was not. Each group was then electrically stimulated for a single time period (0, 5, 30, 60, or 180 min) before measuring jumping muscle lactate, a characteristic of fast-twitch type fibers. At each time point, CES led to a significantly reduced jumping muscle lactate concentration. Based on similar short-term CES mammalian studies, the reduction in lactate production was most likely due to a reduced reliance on anaerobic metabolism. Thus, longer stimulation periods should result in greater aerobic enzymatic activities, altered myosin ATPase, and shift fiber types. This is the first study to use electrical stimulation to explore insect muscle plasticity and our results show that grasshopper jumping muscle responds similarly to mammalian muscle.

Deep muscle-proteomic analysis of freeze-dried human muscle biopsies reveals fiber type-specific adaptations to exercise training.

Skeletal muscle conveys several of the health-promoting effects of exercise; yet the underlying mechanisms are not fully elucidated. Studying skeletal muscle is challenging due to its different fiber types and the presence of non-muscle cells. This can be circumvented by isolation of single muscle fibers. Here, we develop a workflow enabling proteomics analysis of pools of isolated muscle fibers from freeze-dried human muscle biopsies. We identify more than 4000 proteins in slow- and fast-twitch muscle fibers. Exercise training alters expression of 237 and 172 proteins in slow- and fast-twitch muscle fibers, respectively. Interestingly, expression levels of secreted proteins and proteins involved in transcription, mitochondrial metabolism, Ca2+ signaling, and fat and glucose metabolism adapts to training in a fiber type-specific manner. Our data provide a resource to elucidate molecular mechanisms underlying muscle function and health, and our workflow allows fiber type-specific proteomic analyses of snap-frozen non-embedded human muscle biopsies.

Puerarin ameliorates skeletal muscle wasting and fiber type transformation in STZ-induced type 1 diabetic rats.

Puerarin is an isoflavonoid extracted from Pueraria lobate with extensive pharmacological effects in traditional Chinese medicine. The evidence implicates that puerarin mitigates hyperglycemia and various relevant complications. Here, the effect of puerarin on skeletal muscle wasting induced by type 1 diabetes (T1D) was explored. Streptozotocin (STZ)-induced T1D male Sprague Dawley (SD) rats were used in this study. Muscle strength, weight and size were measured. L6 rat skeletal muscle cells were applied for in vitro study. Our results showed that eight-week oral puerarin administration (100 mg/kg) increased muscle strengths and weights accompanied by enhanced skeletal muscle cross-sectional areas in diabetic rats. Simultaneously, puerarin also reduced expressions of several muscle wasting marker genes including F-box only protein 32 (Atrogin-1) and muscle-specific RING-finger 1 (Murf-1) in diabetic group both in vitro and in vivo. Transformation from type I fibers (slow muscle) to type II fibers (fast muscle) were also observed under puerarin administration in diabetic rats. Puerarin promoted Akt/mTOR while inhibited LC3/p62 signaling pathway in skeletal muscle cells. In conclusion, our study showed that puerarin mitigated skeletal muscle wasting in T1D rats and closely related with Akt/mTOR activation and autophagy inhibition. Whether this effect in murine applies to humans remains to be determined.

Differential effects of pre-exercise on cancer cachexia-induced muscle atrophy in fast- and slow-twitch muscles.

We hypothesized that pre-exercise may effectively prevent cancer cachexia-induced muscle atrophy in both fast- and slow-twitch muscle types. Additionally, the fast-twitch muscle may be more affected by cancer cachexia than slow-twitch muscle. This study aimed to evaluate the effects of pre-exercise on cancer cachexia-induced atrophy and on atrophy in fast- and slow-twitch muscles. Twelve male Wistar rats were randomly divided into sedentary and exercise groups, and another 24 rats were randomly divided into control, pre-exercise, cancer cachexia induced by intraperitoneal injections of ascites hepatoma AH130 cells, and pre-exercise plus cancer cachexia groups. We analyzed changes in muscle mass and in gene and protein expression levels of major regulators and indicators of muscle protein degradation and synthesis pathways, angiogenic factors, and mitochondrial function in both the plantaris and soleus muscles. Pre-exercise inhibited muscle mass loss, rescued protein synthesis, prevented capillary regression, and suppressed hypoxia in the plantaris and soleus muscles. Pre-exercise inhibited mitochondrial dysfunction differently in fast- and slow-twitch muscles. These results suggested that pre-exercise has the potential to inhibit cancer-cachexia-induced muscle atrophy in both fast- and slow-twitch muscles. Furthermore, the different progressions of cancer-cachexia-induced muscle atrophy in fast- and slow-twitch muscles are related to differences in mitochondrial function.

Comparison of Functional Characteristics of Myosin in Fast and Slow Skeletal Muscles.

Myosins of fast and slow skeletal muscles differ by the isoform composition of the heavy and light chains. We compared functional characteristics of myosin from the fast (m. psoas) and slow (m. soleus) muscles of rabbits. The parameters of single actin-myosin interaction were measured in an optical trap, and the characteristics of the Ca2+ regulation of actin-myosin interaction were studied using an in vitro motility assay. The duration of interaction of myosin from the fast muscle with actin was shorter and the filament sliding velocity over this myosin was higher than the corresponding parameters for myosin from the slow muscle. The dependence pCa-velocity for myosin from the fast muscle was less sensitive to Ca2+ than that of slow muscle myosin. Thus, functional properties of myosin determine not only mechanical and kinetic characteristics of muscle contraction, but also the peculiarities of its Ca2+ regulation.

An exploratory study of contractile force production in muscle fibers from patients with inflammatory myopathies.

INTRODUCTION: The mechanism by which weakness develops in idiopathic inflammatory myopathies (IIMs) is still unclear. In this study we investigated the maximum force of single muscle fibers from patients with IIMs. METHODS: Permeabilized single muscle fibers from patients with IIMs and healthy controls were subjected to contractility measurements. Maximum force and specific force production (maximum force normalized to fiber size) and fiber type were determined for each isolated fiber. RESULTS: A total of 178 fibers were studied from five patients with IIMs and 95 fibers from four controls. Specific force production was significantly lower in the IIM group for all fiber types. DISCUSSION: The findings from this exploratory study suggest that weakness in IIMs may, in part, be caused by dysfunction of the contractile apparatus. These findings provide a basis for further studies into the mechanisms underlying weakness in IIMs.

Cell fusion is differentially regulated in zebrafish post-embryonic slow and fast muscle.

Skeletal muscle fusion occurs during development, growth, and regeneration. To investigate how muscle fusion compares among different muscle cell types and developmental stages, we studied muscle cell fusion over time in wild-type, myomaker (mymk), and jam2a mutant zebrafish. Using live imaging, we show that embryonic myoblast elongation and fusion correlate tightly with slow muscle cell migration. In wild-type embryos, only fast muscle fibers are multinucleate, consistent with previous work showing that the cell fusion regulator gene mymk is specifically expressed throughout the embryonic fast muscle domain. However, by 3 weeks post-fertilization, slow muscle fibers also become multinucleate. At this late-larval stage, mymk is not expressed in muscle fibers, but is expressed in small cells near muscle fibers. Although previous work showed that both mymk and jam2a are required for embryonic fast muscle cell fusion, we observe that muscle force and function is almost normal in mymk and jam2a mutant embryos, despite the lack of fast muscle multinucleation. We show that genetic requirements change post-embryonically, with jam2a becoming much less important by late-larval stages and mymk now required for muscle fusion and growth in both fast and slow muscle cell types. Correspondingly, adult mymk mutants perform poorly in sprint and endurance tests compared to wild-type and jam2a mutants. We show that adult mymk mutant muscle contains small mononucleate myofibers with average myonuclear domain size equivalent to that in wild type adults. The mymk mutant fibers have decreased Laminin expression and increased numbers of Pax7-positive cells, suggesting that impaired fiber growth and active regeneration contribute to the muscle phenotype. Our findings identify several aspects of muscle fusion that change with time in slow and fast fibers as zebrafish develop beyond embryonic stages.

Synergist ablation-induced hypertrophy occurs more rapidly in the plantaris than soleus muscle in rats due to different molecular mechanisms.

We examined molecular mechanisms that were altered during rapid soleus (type I fiber-dominant) and plantaris (type II fiber-dominant) hypertrophy in rats. Twelve Wistar rats (3.5 mo old; 6 female, 6 male) were subjected to surgical right-leg soleus and plantaris dual overload [synergist ablation (SA)], and sham surgeries were performed on left legs (CTL). At 14 days after surgery, the muscles were dissected. Plantaris mass was 27% greater in the SA than CTL leg (P < 0.001), soleus mass was 13% greater in the SA than CTL leg (P < 0.001), and plantaris mass was higher than soleus mass in the SA leg (P = 0.001). Plantaris total RNA concentrations and estimated total RNA levels (suggestive of ribosome density) were 19% and 47% greater in the SA than CTL leg (P < 0.05), protein synthesis levels were 64% greater in the SA than CTL leg (P = 0.038), and satellite cell number per fiber was 60% greater in the SA than CTL leg (P = 0.003); no differences in these metrics were observed between soleus SA and CTL legs. Plantaris, as well as soleus, 20S proteasome activity was lower in the SA than CTL leg (P < 0.05), although the degree of downregulation was greater in the plantaris than soleus muscle (-63% vs. -20%, P = 0.001). These data suggest that early-phase plantaris hypertrophy occurs more rapidly than soleus hypertrophy, which coincided with greater increases in ribosome biogenesis, protein synthesis, and satellite cell density, as well as greater decrements in 20S proteasome activity, in the plantaris muscle.

SCD1 regulates the AMPK/SIRT1 pathway and histone acetylation through changes in adenine nucleotide metabolism in skeletal muscle.

Stearoyl-CoA desaturase (SCD) is a rate-limiting enzyme that catalyzes the synthesis of monounsaturated fatty acids. It plays an important role in regulating skeletal muscle metabolism. Lack of the SCD1 gene increases the rate of fatty acid ß-oxidation through activation of the AMP-activated protein kinase (AMPK) pathway and the upregulation of genes that are related to fatty acid oxidation. The mechanism of AMPK activation under conditions of SCD1 deficiency has been unclear. In the present study, we found that the ablation/inhibition of SCD1 led to AMPK activation in skeletal muscle through an increase in AMP levels whereas muscle-specific SCD1 overexpression decreased both AMPK phosphorylation and the adenosine monophosphate/adenosine triphosphate (AMP/ATP) ratio. Changes in AMPK phosphorylation that were caused by SCD1 down- and upregulation affected NAD+ levels following changes in NAD+ -dependent deacetylase sirtuin-1 (SIRT1) activity and histone 3 (H3K9) acetylation and methylation status. Moreover, mice with muscle-targeted overexpression of SCD1 were more susceptible to high-fat diet-induced lipid accumulation and the development of insulin resistance compared with wild-type mice. These data show that SCD1 is involved in nucleotide (ATP and NAD+ ) metabolism and suggest that the SCD1-dependent regulation of muscle steatosis and insulin sensitivity are mediated by cooperation between AMPK- and SIRT1-regulated pathways. Altogether, the present study reveals a novel mechanism that links SCD1 with the maintenance of metabolic homeostasis and insulin sensitivity in skeletal muscle.