Evaluating the Bioenergetics Health Index Ratio in Leigh Syndrome Fibroblasts to Understand Disease Severity

Abstract

Several pediatric mitochondrial disorders, including Leigh syndrome (LS), impact mitochondrial (mt) genetics, development, and metabolism, leading to complex pathologies and energy failure. The extent to which pathogenic mtDNA variants regulate disease severity in LS is currently not well understood. To better understand this relationship, we computed a glycolytic bioenergetics health index (BHI) for measuring mitochondrial dysfunction in LS patient fibroblast cells harboring varying percentages of pathogenic mutant mtDNA (T8993G, T9185C) exhibiting deficiency in complex V or complex I (T10158C, T12706C). A high percentage (>90%) of pathogenic mtDNA in cells affecting complex V and a low percentage (<39%) of pathogenic mtDNA in cells affecting complex I was quantified. Levels of defective enzyme activities of the electron transport chain correlated with the percentage of pathogenic mtDNA. Subsequent bioenergetics assays showed cell lines relied on both OXPHOS and glycolysis for meeting energy requirements. Results suggest that whereas the precise mechanism of LS has not been elucidated, a multi-pronged approach taking into consideration the specific pathogenic mtDNA variant, glycolytic BHI, and the composite BHI (average ratio of oxphos to glycolysis) can aid in better understanding the factors influencing disease severity in LS.

Keywords: bioenergetics health index; glycolysis; leigh syndrome; mitochondrial disorders; mitochondrial respiration.

Figure 1

Mitochondrial respiratory profile of CTL BJ-FB and five LS fibroblast cell lines. (a) Scheme of expected oxygen consumption rate (OCR) under basal conditions, (b) representative OCR profile of BJ-FB, (c) representative OCR profile of diseased SBG1-FB (MT-ATP6-T8993G), SBG2-FB (MT-ATP6-T8993G), SBG3-FB (MT-ATP6-T9185C), SBG4-FB (MT-ND3-T10158C) and SBG5-FB (MT-ND5-T12706C) cell lines showing (d) basal respiration, (e) maximal respiration (f) non- mitochondrial respiration after Rot/AA injection proton leak, (g) proton leak (h) spare respiratory capacity, (i) coupling efficiency. All parameters are in pmol/min/1000 cells. Data are mean +/− SD. Experiments were repeated at least three times on different days under the same conditions. * p < 0.05 ** p < 0.01 **** p < 0.0001. Comparative analyses for all diseased (SBG1-5) FBs were conducted with the healthy control BJ-FB line.

Figure 2

Glycolytic profile of CTL BJ-FB and five LS fibroblast cell lines. (a) a real-time profile shows the glycolytic acidification; PER of BJ-FB and diseased SBG1-FB (MT-ATP6-T8993G), SBG2-FB (MT-ATP6-T8993G), SBG3-FB (MT-ATP6-T9185C), SBG4-FB (MT-ND3-T10158C), and SBG5-FB (MT-ND5-T12706C) cell lines showing (b) basal glycolysis (c) glycolytic capacity after ETC blocking using Rot/AA (d) post 2-DG (non-glycolytic acidification). Data are shown in pmol H +/min/1000 cells as mean +/− SD. Experiments were repeated at least three times on different days under the same conditions. * p < 0.05 **** p < 0.0001. Comparative analyses for all diseased (SBG1-5) FBs were conducted with the healthy control BJ-FB line. PER: proton efflux rate.

Figure 3

Production of ATP in BJ-FB and five LS fibroblast cell lines. The LS lines are SBG1-FB (MT-ATP6-T8993G), SBG2-FB (MT-ATP6-T8993G), SBG3-FB (MT-ATP6-T9185C), SBG4-FB (MT-ND3-T10158C), and SBG5-FB (MT-ND5-T12706C), impacting the function of ATP synthase or NADH dehydrogenase. (a) Mitochondrial ATP rate and (b) Glyco-ATP rate. Data are shown in pmol H +/min/1000 cells as mean +/− SD. Experiments were repeated at least three times on different days under the same conditions. **** p < 0.0001. Comparative analyses for all diseased (SBG1-5) FBs were conducted with the healthy control BJ-FB line. OCR: oxygen consumption rate. PER: proton efflux rate.

Figure 4

Composite Bioenergetic Health Index (BHI) ratio for BJ-FB and five LS fibroblast cell lines. The LS lines are SBG1-FB (MT-ATP6-T8993G), SBG2-FB (MT-ATP6-T8993G), SBG3-FB (MT-ATP6-T9185C), SBG4-FB (MT-ND3-T10158C), and SBG5-FB (MT-ND5-T12706C) impacting the function of ATP synthase or NADH dehydrogenase. (a) The formula used to calculate the MitoBHI and GlycoBHI (b) Mito-BHI values were quantitated based on four bioenergetic parameters: mitoATP production, spare reserve capacity, proton leak and non-mitochondrial respiration. Glyco-BHI values were quantitated based on four glycolytic parameters: Basal glycolysis (Basal PER), compensatory glycolysis, mitochondrial acidification (MitoPER), and post 2-DG acidification. ** p < 0.01 **** p < 0.0001.

Figure 5

A model for predicting disease severity in LS. Our results indicate that during stress triggered by specific pathogenic mtDNA variants or other factors, cells SBG4-FB (MT-ND3-T10158C) with high spare reserve capacity (SRC), low heteroplasmy, and high composite BHI ratio exhibit delayed onset and mild clinical symptoms. However, as SRC and composite BHI ratio decreases, cells SBG5-FB (MT-ND5-T12706C) are unable to handle stress and exhibit early-onset and severe clinical symptoms despite low heteroplasmy levels. Whereas cells carrying pathogenic disease mtDNA variants in ATP6 gene SBG1-FB (MT-ATP6-T8993G), SBG2-FB (MT-ATP6- T8993G), SBG3-FB (MT-ATP6-T9185C) exhibit very high heteroplasmy levels, lower composite BHI ratio when compared with control BJ-FB, and can be grouped as ‘intermediate’ in disease severity.