Mito-FUNCAT-FACS reveals cellular heterogeneity in mitochondrial translation.

Mitochondria possess their own genome that encodes components of oxidative phosphorylation (OXPHOS) complexes, and mitochondrial ribosomes within the organelle translate the mRNAs expressed from the mitochondrial genome. Given the differential OXPHOS activity observed in diverse cell types, cell growth conditions, and other circumstances, cellular heterogeneity in mitochondrial translation can be expected. Although individual protein products translated in mitochondria have been monitored, the lack of techniques that address the variation in overall mitochondrial protein synthesis in cell populations poses analytic challenges. Here, we adapted mitochondrial-specific fluorescent noncanonical amino acid tagging (FUNCAT) for use with fluorescence-activated cell sorting (FACS) and developed mito-FUNCAT-FACS. The click chemistry-compatible methionine analog L-homopropargylglycine (HPG) enabled the metabolic labeling of newly synthesized proteins. In the presence of cytosolic translation inhibitors, HPG was selectively incorporated into mitochondrial nascent proteins and conjugated to fluorophores via the click reaction (mito-FUNCAT). The application of in situ mito-FUNCAT to flow cytometry allowed us to separate changes in net mitochondrial translation activity from those of the organelle mass and detect variations in mitochondrial translation in cancer cells. Our approach provides a useful methodology for examining mitochondrial protein synthesis in individual cells.

Transcutaneous application of carbon dioxide improves contractures after immobilization of rat knee joint.

OBJECTIVE: Joint contractures are a major complication following joint immobilization. However, no fully effective treatment has yet been found. Recently, carbon dioxide (CO2) therapy was developed and verified this therapeutic application in various disorders. We aimed to verify the efficacy of transcutaneous CO2 therapy for immobilization-induced joint contracture. METHOD: Twenty-two Wistar rats were randomly assigned to three groups: caged control, those untreated after joint immobilization, and those treated after joint immobilization. The rats were treated with CO2 for 20 min once a daily either during immobilization, (prevention) or during remobilization after immobilization (treatment). Knee extension motion was measured with a goniometer, and the muscular and articular factors responsible for contractures were calculated. We evaluated muscle fibrosis, fibrosis-related genes (collagen Type 1α1 and TGF-ß1) in muscles, synovial intima's length, and fibrosis-related proteins (Type I collagen and TGF-ß1) in the joint capsules. RESULTS: CO2 therapy for prevention and treatment improved the knee extension motion. Muscular and articular factors decreased in rats of the treatment group. The muscular fibrosis of treated rats decreased in the treatment group. Although CO2 therapy did not repress the increased expression of collagen Type 1α1, the therapy decreased the expression of TGF-ß1 in the treatment group. CO2 therapy for treatment improved the shortening of the synovial membrane after immobilization and decreased the immunolabeling of TGF-ß1 in the joint capsules. CONCLUSIONS: CO2 therapy may prevent and treat contractures after joint immobilization, and appears to be more effective as a treatment strategy for the deterioration of contractures during remobilization.

Effects of hypertonia on contracture development in rat spinal cord injury.

STUDY DESIGN: Experimental animal study. OBJECTIVES: Spastic hypertonia is originally believed to cause contractures from clinical observations. Botulinum toxin is effective for the treatment of spasticity and is widely used in patients who have joints with contractures. Using an established rat model with knee contractures after spinal cord injuries, we aimed to verify whether hypertonia contributes to contracture development, and the botulinum toxin improves structural changes in muscles and joint components responsible for contractures. SETTING: University laboratory in Japan. METHODS: To evaluate the effect of hypertonia on contracture development, the rats received botulinum toxin injections after spinal cord injuries. Knee extension motion was measured with a goniometer applying a standardized torque under anesthesia, and the contribution by muscle or non-muscle structures to contractures were calculated by measuring joint motion before and after the myotomies. We quantitatively measured the muscle atrophy, muscle fibrosis, and synovial intima length. RESULTS: Botulinum toxin injections significantly improved contractures, whereas did not completely prevent contracture development. Botulinum toxin was effective in improving the muscular factor, but little difference in the articular factor. Spinal cord injuries induced muscle atrophy, and botulinum toxin significantly accelerated muscle atrophy and fibrosis. The synovial intima length decreased significantly after spinal cord injuries, and botulinum toxin did not improve this shortening. CONCLUSIONS: This animal study provides new evidence that hypertonia is not the sole cause rather is the partial contributor of contractures after spinal cord injuries. Furthermore, botulinum toxin has adverse effects in the muscle.