Accelerometry predicts muscle ultrastructure and flight capabilities in a wild bird

ABSTRACTMuscle ultrastructure is closely linked with athletic performance in humans and lab animals, and presumably plays an important role in the movement ecology of wild animals. Movement is critical for wild animals to forage, escape predators and reproduce. However, little evidence directly links muscle condition to locomotion in the wild. We used GPS-accelerometers to examine flight behaviour and muscle biopsies to assess muscle ultrastructure in breeding black-legged kittiwakes (Rissa tridactyla). Biopsied kittiwakes showed similar reproductive success and subsequent over-winter survival to non-biopsied kittiwakes, suggesting that our study method did not greatly impact foraging ability. Muscle fibre diameter was negatively associated with wing beat frequency, likely because larger muscle fibres facilitate powered flight. The number of nuclei per fibre was positively associated with average air speed, likely because higher power output needed by faster-flying birds required plasticity for muscle fibre recruitment. These results suggest the potential for flight behaviour to predict muscle ultrastructure.

Characterization of Hspb8 in Zebrafish

Hspb8 is a member of the small heat shock protein (sHSP) family. Its expression is known to be upregulated under heat shock. This protein interacts with different partners and can, therefore, be involved in various processes relevant to tissue integrity and functioning. In humans, mutations in the gene encoding Hspb8 can lead to the development of various diseases such as myopathies and neuropathies. In our study, we aimed to perform an in-depth characterization of zebrafish Hspb8 during zebrafish development. We applied techniques such as RT-qPCR, Western blot, immunofluorescence, co-immunoprecipitation, LC-MS, and morpholino-mediated knockdown. We broadened the knowledge regarding zebrafish hspb8 expression during development under normal and heat shock conditions as well as its tissue- and subcellular-specific localization. A co-IP analysis allowed us to conclude that zebrafish Hspb8 can interact with proteins such as Bag3 and Hsc70, which are crucial for formation of an autophagy-inducing complex. We also demonstrated that hspb8 morpholino-mediated knockdown has an impact on zebrafish embryos’ morphology, muscle ultrastructure, and motility behavior. Our research provides a valuable resource for the potential use of the zebrafish as a model for studying pathological conditions associated with hspb8 disorders.

Consequences of being phenotypically mismatched with the environment: rapid muscle ultrastructural changes in cold-shocked black-capped chickadees (Poecile atricapillus)

Phenotypic flexibility has received considerable attention in the last decade; however, whereas many studies have reported amplitude of variation in phenotypic traits, much less attention has focused on the rate at which traits can adjust in response to sudden changes in the environment. We investigated whole animal and muscle phenotypic changes occurring in black-capped chickadees ( Poecile atricapillus) acclimated to cold (−5°C) and warm (20°C) temperatures in the first 3 h following a 15°C temperature drop (over 3 h). Before the temperature change, cold-acclimated birds were consuming 95% more food, were carrying twice as much body fat, and had 23% larger pectoralis muscle fiber diameters than individuals kept at 20°C. In the 3 h following the temperature drop, these same birds altered their pectoralis muscle ultrastructure by increasing the number of capillaries per fiber area and the number of nuclei per millimeter of fiber by 22%, consequently leading to a 22% decrease in myonuclear domain (amount of cytoplasm serviced per nucleus), whereas no such changes were observed in the warm-acclimated birds. To our knowledge, this is the first demonstration of such a rapid adjustment in muscle fiber ultrastructure in vertebrates. These results support the hypothesis that chickadees maintaining a cold phenotype are better prepared than warm-phenotype individuals to respond to a sudden decline in temperature, such as what may be experienced in their natural wintering environment.

Reducing dynamin 2 (DNM2) rescues DNM2-related dominant centronuclear myopathy

Centronuclear myopathies (CNM) are a group of severe muscle diseases for which no effective therapy is currently available. We have previously shown that reduction of the large GTPase DNM2 in a mouse model of the X-linked form, due to loss of myotubularin phosphatase MTM1, prevents the development of the skeletal muscle pathophysiology. As DNM2 is mutated in autosomal dominant forms, here we tested whether DNM2 reduction can rescue DNM2-related CNM in a knock-in mouse harboring the p.R465W mutation (Dnm2RW/+) and displaying a mild CNM phenotype similar to patients with the same mutation. A single intramuscular injection of adeno-associated virus-shRNA targeting Dnm2 resulted in reduction in protein levels 5 wk post injection, with a corresponding improvement in muscle mass and fiber size distribution, as well as an improvement in histopathological CNM features. To establish a systemic treatment, weekly i.p. injections of antisense oligonucleotides targeting Dnm2 were administered to Dnm2RW/+mice for 5 wk. While muscle mass, histopathology, and muscle ultrastructure were perturbed in Dnm2RW/+mice compared with wild-type mice, these features were indistinguishable from wild-type mice after reducing DNM2. Therefore, DNM2 knockdown via two different strategies can efficiently correct the myopathy due to DNM2 mutations, and it provides a common therapeutic strategy for several forms of centronuclear myopathy. Furthermore, we provide an example of treating a dominant disease by targeting both alleles, suggesting that this strategy may be applied to other dominant diseases.

PO-125 Fis1 governs normal mitophagy in slow muscle during the low-intensity and long-period exercise

Objective Mitochondrial dynamics include mitochondrial fusion and mitochondrial fission. It has long been widely recognized that Fis1 plays a role in mitochondrial fission in mammals. However, the finding of Dr. Youle’s team suggests that Fis1 may play an important role in mediating normal mitophagy. Both of mitochondrial dynamics and mitophagy are closely related to skeletal muscle homeostasis. Therefore, in this study, Fis1 was specifically knocked out in skeletal muscle in vivo, looking forward to: 1) investigating the relationship between Fis1 and mitochondrial morphology, mitophagy in mouse skeletal muscle. 2) The mechanism of Fis1 in mitochondrial quality in skeletal muscle under exercise stress. So as to we can clarify the molecular mechanism of Fis1 in mediating mitochondrial quality in skeletal muscle, but also expect to provide more theoretical basis for skeletal muscle health and exercise adaptation. Methods We constructed conditional skeletal muscle Fis1 knockout mice (C57BL/6) and littermate control mice through Cre / Loxp technique. The mice were free feeding, drinking and activity during the teat, we only selected male mice for all of the tests. And the genotypes were Fis1FL / FL MCK-Cre + (Fis1KO) and Fis1FL / FL MCK-Cre – (WT). First, we performed endurance test on 10 WT and 10 Fis1KO mice (32-40 weeks, n = 10), then dissected quadriceps, gastrocnemius and soleus (n=4-5) quickly and rapidly frozen in liquid nitrogen, then stored at -80 ° C freezer for testing Fis1 and OXPHOS expression (Western-blot). On the other hand, we selected WT and Fis1KO mice (n=3) to prepare EM samples, so as to observe mitochondrial morphology and muscle ultrastructure. Skeletal muscle (n=3-4) was snap-frozen in isopentane cooled with liquid nitrogen for HE, NADH staining, and observing GFP-LC3 (mitophagy). Base on the exploration of loss of Fis1 without stress, we adopted endurance exhaustive exercise on WT (WT EEE) and Fis1KO mice (Fis1KO EEE) (n=3-4). Mice were acclimated to and trained on a 10o uphill treadmill. Mice were acclimated to and trained on a 10o uphill treadmill (Columbus Instruments) for 2 days. On day 3, mice were subjected to a single bout of running starting at the speed of 10m/min. Forty minutes later, the treadmill speed was increased at a rate of 1m/min every 10 min for a total of 30 min, and then increased at the rate of 1m/min every 5 min until mice were exhausted. Exhaustion was defined as the point at which mice spent more than 5 s on the electric shocker without attempting to resume running even if we used short air puffs and tail tickles with bristle brush. We dissected soleus and gastrocnemius to observe muscle ultrastructure and mitophagy through EM and confocal microscope respectively (same methods as before). At the same time, we used immune-EM to observe autophagosome morphology and LC3 distribution. Results Behavior test on specific knock out skeletal muscle Fis1 mice model We found that loss of Fis1 induced significantly lower performance in treadmill endurance tests than controls (P <0.001). The effect of loss of Fis1 on mitochondrial morphology and function In soleus, knocking out Fis1 caused mitochondrial hyperfusion (mitochondrial size was significantly increased, P = 0.01). In addition, we found more swollen mitochondria in Fis1 knock-out gastrocnemius. On the other hand, compared with the control mice, lack of Fis1 significantly reduced the protein expression of Complex I, Complex II and Complex IV in soleus (P <0.01). As same as before, we also found a significant increase GFP-LC3 (P <0.01) in Fis1KO soleus. 3) Changes of muscle ultrastructure and mitophagy after endurance exhaustive exercise (EEE) First, comparing with the control group, swollen sarcoplasmic reticulum (skeletal muscle endoplasmic reticulum (ER)) and extremely swollen terminal cisternae (TC) were found in Fis1KO soleus and gastrocnemius respectively after endurance exhaustive exercise. We found a significant accumulation of GFP-LC3 (P <0.0001) in Fis1KO soleus compared to the control. However, GFP-LC3 signal still increased (P <0.001) in Fis1KO soleus after exercise compared with that in soleus before exercise. Moreover, we observed a lot of large and irregular autophagosomes appeared in Fis1KO soleus after EEE through immune electron microscope. Conclusions 1) Loss of Fis1 causes a certain degree of mitochondrial hyperfusion, increases mitophagy and significantly decreases mitochondrial function in slow muscle. However, losing Fis1 does not cause obvious alteration on quick muscle and synthetic muscle. Therefore, the absence of Fis1 has a significant effect on mitochondria-rich muscle. 2) Mitochondrial-ER interactions may be involved in the connection of endoplasmic reticulum swelling after endurance exhaustive exercise. 3) Endurance exercise with oxidative phosphorylation aggravate the increase and abnormality of mitophagy caused by the loss of Fis1 in slow muscle, suggesting that Fis1 governs normal mitophagy in slow muscle during the low-intensity and long-period exercise. This phenomenon may be related to the worse performance in treadmill endurance test of Fis1 KO mice.

Effect of Dissection and Reconstruction of Palatal Muscles on Morphological Features and Ultrastructure of the Oral Musculature in Cats

The study was designed to determine the effect of dissection and reconstruction of palatal muscles on muscle morphology in cats. 27 cats were randomly divided into three groups according to the extent of muscle dissection from the palatal midline. All dissections were performed from the posterior border of the hard palate, and the muscles were allowed to reconstruct over time. The morphological features were determined by hematoxylin and eosin staining of tissue sections, and ultrastructure was observed under a transmission electron microscope. As a result, no obvious differences were evident in the morphological features or ultrastructure of animals in the <1/3rd and 1/3rd-2/3rd area groups. In the >2/3rd area group, the muscles fibers were disordered and inflammatory cell infiltration and naïve muscle cells were found at one month after surgery. At the second and third month after surgery, the muscle fibers showed regular alignment, the naïve muscle fibers gradually matured, and the number of infiltrating inflammatory cells decreased. Muscle ultrastructure analysis revealed that myocommata were correctly aligned, and the Z line was more distinct. In conclusion, extensive dissection of palatal muscles does not result in fibrosis. Injury to oral musculature can be repaired and the musculature regenerated over time.