A novel immunofluorescent assay to investigate oxidative phosphorylation deficiency in mitochondrial myopathy: understanding mechanisms and improving diagnosis

Mariana C Rocha,Philip F Dobson,Karolina A Rygiel,Anne Grünewald,Robert W Taylor,Amy Vincent,Doug M Turnbull,John P Grady

doi:10.1038/srep15037

Abstract

Oxidative phosphorylation defects in human tissues are often challenging to quantify due to a mosaic pattern of deficiency. Biochemical assays are difficult to interpret due to the varying enzyme deficiency levels found in individual cells. Histochemical analysis allows semi-quantitative assessment of complex II and complex IV activities, but there is no validated histochemical assay to assess complex I activity which is frequently affected in mitochondrial pathology. To help improve the diagnosis of mitochondrial disease and to study the mechanisms underlying mitochondrial abnormalities in disease, we have developed a quadruple immunofluorescent technique enabling the quantification of key respiratory chain subunits of complexes I and IV, together with an indicator of mitochondrial mass and a cell membrane marker. This assay gives precise and objective quantification of protein abundance in large numbers of individual muscle fibres. By assessing muscle biopsies from subjects with a range of different mitochondrial genetic defects we have demonstrated that specific genotypes exhibit distinct biochemical signatures in muscle, providing evidence for the diagnostic use of the technique, as well as insight into the underlying molecular pathology. Stringent testing for reproducibility and sensitivity confirms the potential value of the technique for mechanistic studies of disease and in the evaluation of therapeutic approaches.

Highlights

Affected in patients with a multisystem phenotype[7,8]
Though valuable for detecting the mosaic oxidative phosphorylation (OXPHOS) deficiency observed in many mitochondrial myopathies, myofibre classification is subjective, qualitative and prone to inter-observer variability[14,15]
Using laminin to define cell boundaries enables automatic quantification of myofibres[29]. We demonstrate that this technique is sensitive and reliable in detecting and quantifying focal OXPHOS deficiency in human muscle, and show that different mitochondrial genotypes – both mtDNA and nuclear DNA driven - exhibit distinctive signatures of mitochondrial dysfunction

Summary

Introduction

Affected in patients with a multisystem phenotype[7,8]. Involvement of mtDNA usually leads to mosaic deficiency of the OXPHOS in muscle; mtDNA is present in multiple copies in cells and mutations are frequently heteroplasmic (mixed mutated and wild-type mtDNA within an individual cell). Current diagnostic algorithms for investigating and diagnosing mitochondrial disease use histochemical and biochemical assessment of OXPHOS activities in clinically-affected tissues[11]. Biochemical assays measure activity of each individual OXPHOS enzyme complex (complex I–V) in homogenised muscle tissue[11]. Whilst they are very useful for assessment of wide-spread mitochondrial defects, they may fail to detect more subtle OXPHOS deficiencies, especially when only a few myofibres are involved. They require substantial quantities of muscle (> 50 mg) which are not always available. To overcome the limitations of currently available methods we have developed an objective and semi-automated technique allowing quantification of mitochondrial dysfunction in individual myofibres

Results

Discussion

Conclusion