Rationale:In skeletal muscles, there are four myofiber types, Types I, IIa, IIx, and IIb, which show different contraction characteristics and have different metabolic statuses. To understand muscle function, it is necessary to analyze myofiber-specific metabolic changes. However, these fibers are heterogeneous and are hard to discriminate by conventional analyses using tissue extracts. In this study, we found myofiber-specific molecules and molecular markers of other cells such as smooth muscle cells, fat cells, and motor neurons, and visualized them within muscle sections. Methods:We used three different muscle tissues, namely extensor digitorum longus, soleus, and gastrocnemius tissues, from ICR mice. After the muscles had been harvested, cross-sections were prepared using a cryostat and analyzed using matrixassisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI), and conventional immunofluorescence imaging.Results: By comparing the MALDI MSI results with the immunofluorescence imaging results, we were able to identify each fiber and cell-specific ion. It was especially important that we could find Type IIa and IIb specific ions, because these were difficult to distinguish. Conclusions:Through MSI analyses, we performed a comprehensive survey to identify cell-and myofiber-specific molecular markers. In conclusion, we assigned muscle fiber Type I, IIa, and IIb-specific molecular ions at m/z 856.6, 872.6, and 683.8, respectively. These molecular markers might be useful for verifying changes that occur due to exercise and/or disease.

Alaska pollack protein (APP) was previously shown to reduce serum triacylglycerol and the atherogenic index and significantly increase gastrocnemius muscle mass in rats. To determine which myofibers are involved in this observed hypertrophy, we stained the gastrocnemius muscle with fast and slow fiber-specific antibodies and measured the muscle fiber diameter. We observed muscle hypertrophy in both the fast and slow fibers of APP-fed rats. Although muscle hypertrophy leads to drastic lipid changes, the amount of lipids did not differ significantly between casein-fed and APP-fed rats. To determine the lipid changes at the molecular species level and their localization, we performed matrix-assisted laser desorption/ ionization mass spectrometry imaging to visualize lipids in the gastrocnemius muscles. We determined that lipid molecules were significantly changed due to APP feeding. Thus, APP feeding changes muscle lipid metabolism, and these metabolic changes might be related to hypertrophy.

The threat of predation is a driving force in the evolution of animals. We have previously reported that Xenopus laevis enhanced their tail muscles and increased their swimming speeds in the presence of Japanese larval salamander predators. Herein, we investigated the induced gene expression changes in the brains of tadpoles under the threat of predation using 3′-tag digital gene expression profiling. We found that many muscle genes were expressed after 24 h of exposure to predation. Ingenuity pathway analysis further showed that after 24 h of a predation threat, various signal transduction genes were stimulated, such as those affecting the actin cytoskeleton and CREB pathways, and that these might increase microtubule dynamics, axonogenesis, cognition, and memory. To verify the increase in microtubule dynamics, DiI was inserted through the tadpole nostrils. Extension of the axons was clearly observed from the nostril to the diencephalon and was significantly increased (P ≤ 0.0001) after 24 h of exposure to predation, compared with that of the control. The dynamic changes in the signal transductions appeared to bring about new connections in the neural networks, as suggested by the microtubule dynamics. These connections may result in improved memory and cognition abilities, and subsequently increase survivability.

Predator-induced adaptive phenotypic plasticity is essential for evolution. However, Xenopus tropicalis tadpoles do not exhibit distinct phenotypes when exposed to new predation threats. Here, we investigated adaptions within their brain. Principal component analysis using morphological parameters indicated that short-term predation threats (24 hr) altered tadpole morphology unlike the control or 5 day-out treatment (exposure to predation for 5 days and then no exposure for 5 days). Whole-brain ingenuity pathway and metabolome analyses revealed that free radicals, superoxide dismutase, glycogenesis, and pyruvate were elevated after 6 hr of predation pressure. Hemoglobin was also synthesized in the brain, forming oxyhemoglobin in the midbrain and hindbrain to reduce radical production. Furthermore, ATP production through glycolysis was promoted, while β-oxidation and the tricarboxylic acid cycle were downregulated. We also predicted increases in microtubule dynamics, neuronal branching, and neuritogenesis. Therefore, X. tropicalis tadpoles can adapt to predation stress through changes within their central nervous system.

Organisms adapt to changes in their environment to survive. The emergence of predators is an example of environmental change, and organisms try to change their external phenotypic systems and physiological mechanisms to adapt to such changes. In general, prey exhibit different phenotypes to predators owing to historically long-term prey-predator interactions. However, when presented with a novel predator, the extent and rate of phenotypic plasticity in prey are largely unknown. Therefore, exploring the physiological adaptive response of organisms to novel predators is a crucial topic in physiology and evolutionary biology. Counterintuitively, Xenopus tropicalis tadpoles do not exhibit distinct external phenotypes when exposed to new predation threats. Accordingly, we examined the brains of X. tropicalis tadpoles to understand their response to novel predation pressure in the absence of apparent external morphological adaptations. Principal component analysis of fifteen external morphological parameters showed that each external morphological site varied nonlinearly with predator exposure time. However, the overall percentage change in principal components during the predation threat (24 h) was shown to significantly (p < 0.05) alter tadpole morphology compared with that during control or 5-day out treatment (5 days of exposure to predation followed by 5 days of no exposure). However, the adaptive strategy of the altered sites was unknown because the changes were not specific to a particular site but were rather nonlinear in various sites. Therefore, RNA-seq, metabolomic, Ingenuity Pathway Analysis, and Kyoto Encyclopedia of Genes and Genomes analyses were performed on the entire brain to investigate physiological changes in the brain, finding that glycolysis-driven ATP production was enhanced and ß-oxidation and the tricarboxylic acid cycle were downregulated in response to predation stress. Superoxide dismutase was upregulated after 6 h of exposure to new predation pressure, and radical production was reduced. Hemoglobin was also increased in the brain, forming oxyhemoglobin, which is known to scavenge hydroxyl radicals in the midbrain and hindbrain. These suggest that X. tropicalis tadpoles do not develop external morphological adaptations that are positively correlated with predation pressure, such as tail elongation, in response to novel predators; however, they improve their brain functionality when exposed to a novel predator.