Polymorphisms in the mitochondrial ribosome recycling factor EF-G2mt/MEF2 compromise cell respiratory function and increase atorvastatin toxicity

Abstract

Mitochondrial translation, essential for synthesis of the electron transport chain complexes in the mitochondria, is governed by nuclear encoded genes. Polymorphisms within these genes are increasingly being implicated in disease and may also trigger adverse drug reactions. Statins, a class of HMG-CoA reductase inhibitors used to treat hypercholesterolemia, are among the most widely prescribed drugs in the world. However, a significant proportion of users suffer side effects of varying severity that commonly affect skeletal muscle. The mitochondria are one of the molecular targets of statins, and these drugs have been known to uncover otherwise silent mitochondrial mutations. Based on yeast genetic studies, we identify the mitochondrial translation factor MEF2 as a mediator of atorvastatin toxicity. The human ortholog of MEF2 is the Elongation Factor Gene (EF-G) 2, which has previously been shown to play a specific role in mitochondrial ribosome recycling. Using small interfering RNA (siRNA) silencing of expression in human cell lines, we demonstrate that the EF-G2mt gene is required for cell growth on galactose medium, signifying an essential role for this gene in aerobic respiration. Furthermore, EF-G2mt silenced cell lines have increased susceptibility to cell death in the presence of atorvastatin. Using yeast as a model, conserved amino acid variants, which arise from non-synonymous single nucleotide polymorphisms (SNPs) in the EF-G2mt gene, were generated in the yeast MEF2 gene. Although these mutations do not produce an obvious growth phenotype, three mutations reveal an atorvastatin-sensitive phenotype and further analysis uncovers a decreased respiratory capacity. These findings constitute the first reported phenotype associated with SNPs in the EF-G2mt gene and implicate the human EF-G2mt gene as a pharmacogenetic candidate gene for statin toxicity in humans.

Figure 1. Viability of yeast heterozygous and haploid deletion mutants in atorvastatin after 5 days.

(A) Percentage cell viability of heterozygous deletion mutants in 110 µM atorvastatin relative to viability in the solvent control. (B) Percentage cell viability of haploid deletion mutants in 110 µM atorvastatin relative to viability in the solvent control. Data represent mean ± SEM (n = 3). A one-way ANOVA, followed by a Dunnett's multiple comparison test, was used to compare the mean percentage viability of the mutant strains to that of the wild-type. *P<0.05, ***P<0.001.

Figure 2. Growth of siRNA transfected RD cells in glucose and galactose medium.

At 24 hours post-transfection, cells were seeded into wells of a 96-well plate in DMEM medium containing 10% fetal bovine serum and either 4.5 g/L glucose (left panel) or 4.5 g/L galactose (right panel). Cell proliferation was determined daily using a luminescent cell viability assay. An untransfected cell line and the rho0 cell line were included as controls. Cell proliferation is shown as a percentage of the maximum cell growth (100%) of the untransfected control. Error bars represent the mean ± SEM for three independent measurements.

Figure 3. EF-G2mt protein variants.

Alignment of the protein amino acid sequence of the human EF-G2mt protein with the yeast Mef2 protein. Dark shaded areas represent conserved amino acid residues and grey shaded areas represent semi-conserved residues. EF-G2mt SNPs that are semiconserved in yeast MEF2 are shown in italics and fully conserved SNPs are depicted in bold. The five alleles selected for functional characterisation are outlined. The five EF-G2mt protein domains are represented below the alignment. Global alignment of protein sequences was performed using Lalign and the BioEdit sequence alignment editor was used to generate the graphical representation.

Figure 4. Functional characterisation of EF-G2mt equivalent SNPs in the yeast MEF2 gene.

(A) Growth of wild-type, mef2 deletion strain and mef2 mutants in yeast medium containing either glucose as the carbon source or the non-fermentable carbon source glycerol. Cells from exponentially growing cultures were serially diluted and 5 µl of each dilution spotted onto each plate. Growth was assessed after 72 hours incubation at 30°C. (B) Percentage cell viability of yeast mef2 mutants in 110 µM atorvastatin relative to viability of the wild-type strain following exposure to atorvastatin for 5 days. Data represent mean ± SEM (n = 3). A one-way ANOVA followed by a Dunnett's multiple comparison test was used to compare the mean percentage viability of the mef2 variants to that of the wild-type. ***P<0.001.

Figure 5. Oxygen consumption rates of mef2 mutant cultures.

Figure depicts the rate of oxygen consumption per minute per 30 mL of yeast culture at an OD₆₀₀ of 0.2. Data represent mean ± SEM (n = 3). A one-way ANOVA followed by a Dunnett's multiple comparison test was used to compare the mean oxygen consumption rate of mef2 variants to that of the wild-type. ***P<0.001.

Figure 6. Visualisation of mitochondrial membrane potential.

Cells were stained with MitoTracker Red CMXRos and observed using a laser scanning confocal microscope. To better visualise mitochondrial structure within the mef2 deletant, cells were stained with a 10× concentration of MitoTracker Red. Scale bar represents 5 µm.

Figure 7. In silico model of the human EF-G2mt protein.

(A) Model depicting four of the five amino acid variants that were functionally characterised in this study. (B) Five newly discovered variants that have yet to be functionally characterised. Helices are shown as ribbons, beta-sheets are depicted as flat broad arrows and loops and coils appear as thin tubes. The five protein domains of the EF-G2mt protein are distinguished by different colours. Domain I, the GTP binding domain is shown in blue, domain II is purple, domain III is orange, domain IV is cyan and domain V is green. Model was visualised using the Visual Molecular Dynamics program (VMD), version 1.8.7 (University of Illinois).