2.12. NAD+ metabolism and mitochondrial in situ analyses

NAD⁺ and NADH were measured from gastrocnemius muscle (n = 6 per group) by using slightly modified conventional colorimetric enzymatic assays (see NADmed™ https://www.helsinki.fi/en/researchgroups/mitochondrial-medicine/nadmed). The activity of main cellular NAD⁺ consumers, poly (ADP-ribose) polymerases (PARPs) was determined from gastrocnemius muscle with the HT Colorimetric PARP/Apoptosis Assay Kit (Trevigen, Inc., Gaithersburg, MD, USA) according to manufacturer's instructions (n = 5–6 per group).

For mitochondrial activity in situ analysis, serial cross-sections (8 μm) from TA muscle were cut on a cryomicrotome (at −25 °C; experiment 1, encompassing mice from CTRL, C26 + PBS and C26 + sACVR/c groups). Mitochondrial activity was investigated by assessing the in situ activity of succinate dehydrogenase (SDH, complex II) and cytochrome c oxidase (COX, complex IV) from frozen TA muscle sections by using histochemistry. Cross-sectional images were digitally captured by using a Zeiss Axioplan 2 light microscope (Carl Zeiss AG, Germany) with a ProgRes C7 camera and ProgRes CapturePro 2.9.0.1 software (Jenoptik AG, Germany). Two fields-of-view per muscle cross-section were captured with a 5× magnification. A sample was excluded if cutting of the cross-section was unsuccessful (resulted in n = 5–7 per group). The COX and SDH in situ activities were analysed with Fiji/ImageJ (NIH, Bethesda, MD, USA) plugin Trainable Weka Segmentation [26]. Image resolution was reduced to 1/4 of the original because of the computational power required for analysis. Regions of interest were determined separately for each image and, after splitting channels, applied to green channel images to obtain optical density values of different fibre types. Optical density was assessed from all fibres and from high COX and high SDH fibres. Muscle distribution of mitochondria-rich, high SDH and COX fibres was calculated by using the ImageJ plugin Cell Counter and represented as percentage of high optical density fibres from all fibres.