Characteristics of the mitochondrial network during the process of skeletal muscle regeneration using photothermal microscopy

Background: Skeletal muscle mitochondria repeatedly divide and fuse in response to changing energy supply and demand. These dynamic processes of mitochondrial biogenesis form a catenated network of interconnected mitochondria. However, the characteristics of this behavior during skeletal muscle myogenesis and regeneration are unknown. We have previously developed a photothermal microscopy (PTM) method for intramyocyte localization of cytochrome c using histochemical sections for bright field observation. As cytochrome c is a heme protein bound to the inner mitochondrial membrane PTM facilitates resolution of whole-cell mitochondrial remodeling during muscle regeneration. Purpose: This investigation used PTM to resolve structural changes in the mitochondrial network of gastrocnemius myocytes during regeneration following eccentric contractions (ECCs) induced damage. The reliability of this PTM mitochondrial network characterization was assessed via parallel analysis of PTM and transmission electron microscopy (TEM) images. METHOD: Under anesthesia, 300 ECCs were applied to the gastrocnemius of male Wistar rats (13 weeks old) using electrical stimulation and motor drive-induced muscle lengthening. Seven days into post-ECC recovery (i.e., ECC 7-day), the muscles were perfused-fixed in situ, dissected and embedded in Epon and compared with an ECC non-load control group (CONT). Transverse sections were prepared for optical (1 μm) and electron (1 nm) microscopy. The volume density of mitochondria was evaluated with respect to the distance from the nucleus using PTM and TEM and determination of the network index Form Factor, (FF). PTM used a laser with 515 and 638 nm wavelengths for the PT pump and probe beams, respectively. Results: Seven days after ECC, many regenerated muscle fibers were observed with small diameters and central nuclei, characteristic of the recovery process after muscle damage. Using PTM, across the entire myocyte, a contiguous network of mitochondria, arranged predominantly along the myofibrils, was observed in CONT muscle. In marked contrast, the network index FF was significantly and substantially lower in regenerating muscle (CONT: 15.5 ± 1.8, ECC: 0.6 ± 0.0, p<0.001) evidencing a far more randomized morphology in regenerating (ECC 7-day) than normal (CONT) muscle. Furthermore, in regenerating muscle, mitochondria were aggregated strongly around the central nuclei. Similarly, the mitochondrial volume density in the close proximity of the central nuclei was significantly higher in TEM observations. Specifically, compared to normal muscle, the volume density of mitochondria was significantly higher in the region within 0.1 μm from the nucleus in regenerating muscle (CONT: 4.1±0.2%, ECC: 9.0±0.2%, p<0.0001) and 0.2 μm (CONT: 1.4±0.0%, ECC: 7.6±0.2%, p<0.0001). Conclusion: Muscle histochemical imaging with PTM allows high resolution mitochondrial quantification. During muscle regeneration 7 days post-ECC, PTM analyses revealed a characteristic morphology in which the mitochondria were concentrated preferentially around the nucleus rather than forming a diffuse interconnected network distributed across the entire myocyte characteristic of CONT. This work was supported by JSPS KAKENHI Grant Numbers 23K18417, 24K02827. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.