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Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy
Published on: February 7, 2015
Quantifying variability of mitochondrial markers in m3243A > G myopathy
Tiago M Bernardino Gomes1,2, Jordan B Childs3, Valeria Di Leo3,4
1Mitochondrial Research Group, Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK. tiago.gomes@newcastle.ac.uk.
Abstract:
Myopathy is a prevalent and disabling feature of mitochondrial disease, in which skeletal muscle accumulates fibres with mitochondrial dysfunction in a variable mosaic pattern. This intra-individual spatial heterogeneity, a key consideration in longitudinal assessments, remains largely uncharacterised, hindering mechanistic studies and clinical trials by obscuring or confounding findings. We quantified this variability in m.3243 A > G-related myopathy, a leading cause of adult mitochondrial disease. Post-mortem biopsies from quadriceps femoris and tibialis anterior muscles of four patients were analysed for single-fibre deficiency in oxidative phosphorylation (OXPHOS) complex I and IV, while homogenate mitochondrial DNA (mtDNA) copy number and m.3243 A > G heteroplasmy were respectively determined by quantitative PCR and pyrosequencing. Bootstrapped combinatorial analyses established thresholds for minimum meaningful change above the 97.5th percentile, while accounting for anatomical biopsy distancing. Spatial variability in the proportion of OXPHOS-deficient fibres increased with distancing; within the same muscle, this threshold was 13.8% for NDUFB8 and 9.8% for MT-CO1. Variability in mtDNA copy number modestly increased with distance, while m.3243 A > G heteroplasmy remained largely stable, with within-muscle thresholds of 1,136 copies per nucleus and 8.2%, respectively. These findings provide assay-specific thresholds and offer mechanistic and translational insights for trial design, patient monitoring, and reliable detection of disease progression or therapeutic response.
Insights
Mitochondrial myopathy shows significant spatial variability in muscle fibers. This study quantifies this heterogeneity, providing crucial thresholds for assessing disease progression and therapeutic responses in clinical trials.
Area of Science:
- Mitochondrial Myopathy Research
- Skeletal Muscle Physiology
- Genetic Disease Heterogeneity
Background:
- Myopathy is a common, debilitating symptom of mitochondrial disease.
- Skeletal muscle exhibits mitochondrial dysfunction in a mosaic pattern, complicating assessments.
- Spatial heterogeneity in mitochondrial myopathy is poorly understood, hindering research and clinical trials.
Purpose of the Study:
- To quantify spatial variability in m.3243A>G-related mitochondrial myopathy.
- To establish thresholds for meaningful change in mitochondrial dysfunction markers.
- To provide insights for clinical trial design and patient monitoring.
Main Methods:
- Analysis of post-mortem muscle biopsies (quadriceps femoris, tibialis anterior) from four patients.
- Quantification of single-fiber oxidative phosphorylation (OXPHOS) complex I and IV deficiency.
- Measurement of mitochondrial DNA (mtDNA) copy number and m.3243A>G heteroplasmy using qPCR and pyrosequencing.
- Application of bootstrapped combinatorial analyses to determine variability thresholds accounting for biopsy distance.
Main Results:
- Spatial variability in OXPHOS-deficient fibers increased with muscle biopsy distance.
- Within-muscle thresholds for fiber deficiency were 13.8% (NDUFB8) and 9.8% (MT-CO1).
- mtDNA copy number variability increased modestly with distance; m.3243A>G heteroplasmy remained stable with thresholds of 1,136 copies/nucleus and 8.2%.
Conclusions:
- Established assay-specific thresholds for meaningful change in mitochondrial myopathy markers.
- Findings offer mechanistic insights into spatial heterogeneity of mitochondrial dysfunction.
- Provides translational value for designing clinical trials, monitoring patients, and detecting disease progression or treatment effects.
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