Characterization of myofiber‐type‐specific molecules using mass spectrometry imaging

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 matrix‐assisted 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.