Mitochondrial dysfunction is well described in many chronic illnesses including musculoskeletal, neurodegenerative, and cardiovascular diseases. In this regard, mitochondrial network morphology has been implicated as a biomarker of disease correlating increased mitochondrial fragmentation to impaired cellular function. While advancements in imaging techniques have furthered our understanding of mitochondrial dynamics in live cells, easily accessible approaches for accurate quantification of in situ, whole muscle mitochondria are lacking. PURPOSE: The purpose of this study was to validate a proof-of-concept method capable of quantifying 3D mitochondrial network morphology in whole mount skeletal muscle and then applying it to mitochondrial morphology analysis in multiple cell types: terminal Schwann cell and lymphatic vessel mitochondria networks. HYPOTHESIS: We hypothesized that mitochondrial dysfunction, signified by an increase in dispersed mitochondrial fragments, would coincide with impaired muscle force production in a validated murine model of muscle dystrophy. METHODS: Six-month-old (n=3) adult male DBA/2J (Wild-type, WT) and D2.mdx (MDX) mice were administered a mitochondrial dye (Mitoview Fix 640, Biotium) suspended in saline via intramuscular injection in the right tibialis anterior (TA) muscle. The left TA muscle was isolated for in situ muscle force measurements (Aurora Scientific) using direct electrical stimulation. The dye incubated in vivo for 1 hour prior to experimentation to facilitate uptake in mitochondrial networks. Isolated muscle fibers from the excised TA were fixed, stained with fluorescent Hoechst (Nuclei) and cleared in VisikolTM. 3D confocal Z-stacks (~50 µm) were acquired on a Stellaris 5 White Light Confocal Microscope using Leica LAS_X software (Leica Biosystems) and quantified using machine-learning 3D analysis software (AIVIA). RESULTS: MDX mice had a severe reduction in muscle force compared to WT mice (means ± SE: WT 108 ± 11.44 g; MDX, 56.55 ± 3.3 g; P= 0.01). Analysis of 3D optical sections revealed MDX mice had a relative 2.1-fold increase in small mitochondria fragmentation (WT, 17.2 ± 3.9%; MDX, 26.3 ± 9.7%; P= 0.01). MDX mice exhibited a decrease in large interconnected mitochondrial networks compared to WT mice but did not reach significance (WT, 33.9 ± 6.1%; MDX, 21.1 ± 2.3%; P= 0.11). Correlation analysis demonstrated a strong relationship between relative percentage of large, interconnected mitochondria and peak muscle force production (R2= 0.86, P= 0.02). CONCLUSION: Results support the hypothesis that disruptions in mitochondrial network morphology coincide with reduced muscle function in D2.mdx mice. Moreover, these methods offer a comprehensive and novel approach enabling the quantification of mitochondria network morphology across multiple cell types using standard high-resolution confocal microscopy typically available in university core facilities.



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