Genetic Complementation of ATP Synthase Deficiency Due to Dysfunction of TMEM70 Assembly Factor in Rat

Abstract

Mutations of the TMEM70 gene disrupt the biogenesis of the ATP synthase and represent the most frequent cause of autosomal recessive encephalo-cardio-myopathy with neonatal onset. Patient tissues show isolated defects in the ATP synthase, leading to the impaired mitochondrial synthesis of ATP and insufficient energy provision. In the current study, we tested the efficiency of gene complementation by using a transgenic rescue approach in spontaneously hypertensive rats with the targeted Tmem70 gene (SHR-Tmem70^ko/ko), which leads to embryonic lethality. We generated SHR-Tmem70^ko/ko knockout rats expressing the Tmem70 wild-type transgene (SHR-Tmem70^ko/ko,tg/tg) under the control of the EF-1α universal promoter. Transgenic rescue resulted in viable animals that showed the variable expression of the Tmem70 transgene across the range of tissues and only minor differences in terms of the growth parameters. The TMEM70 protein was restored to 16-49% of the controls in the liver and heart, which was sufficient for the full biochemical complementation of ATP synthase biogenesis as well as for mitochondrial energetic function in the liver. In the heart, we observed partial biochemical complementation, especially in SHR-Tmem70^ko/ko,tg/0 hemizygotes. As a result, this led to a minor impairment in left ventricle function. Overall, the transgenic rescue of Tmem70 in SHR-Tmem70^ko/ko knockout rats resulted in the efficient complementation of ATP synthase deficiency and thus in the successful genetic treatment of an otherwise fatal mitochondrial disorder.

Keywords: ATP synthase deficiency; TMEM70 factor; gene therapy; mitochondria disease; transgenic rescue.

Figure 1

Spontaneously hypertensive rat (SHR)-Tmem70^ko/ko production and Tmem70 transgenic complementation. (A) Generation of SHR-Tmem70^ko/ko rats by ZFN-induced deletion of 131 bp in exon 1. (B) Transgenic rescue by crossing of SHR-Tmem70^ko/wt knockout heterozygotes with SHR-Tmem70^tg/tg transgenic rats expressing wild-type Tmem70 transgene under control of the universal EF-1α promoter. Orange bars (primers A) indicate the position of primers that are specific for transgene construct; blue bars (primers B) represent the position of the primers used for the quantification of the total Tmem70 transcript—endogenous and transgenic. EX—exon, INT—intron, UTR—untranslated region.

Figure 2

Tmem70 transgene expression in SHR rats across tissues and lines. Transgenic Tmem70 mRNA levels were analysed in the intestine, brain, brown adipose tissue (BAT), skeletal muscle, heart, and liver of three animal lines, 130, 126, and 116, and normalized to Hprt expression. Data are mean ± SEM, n = 2–4, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 3

Growth parameters in Tmem70 transgenic rats. Body weight (BW) and heart weight (HW) or liver weight (LW) relative to body weight (LW/BW, HW/BW) were analysed in control SHR rats and in SHR-Tmem70^ko/ko,tg/0 (tg/0) and SHR-Tmem70^ko/ko,tg/tg (tg/tg) rats derived from transgenic lines 130 and 126). Data are mean ± SEM, n = 3–8, *** p ≤ 0.001.

Figure 4

Transgenic complementation of ATP synthase deficiency. (A) WB detection of ATP syn-thase subunits in liver and heart homogenates and (B) quantification of the content of the F1-α, Fo-a, Fo-b, and Fo-c subunits relative to the SDH content (SDHA) in the control SHR rats and in the SHR-Tmem70ko/ko,tg/0 (tg/0) or SHR-Tmem70ko/ko,tg/tg (tg/tg) rats derived from SHR-Tmem70tg/tg trans-genic lines 130 and 126. (C) Tissue Tmem70 mRNA levels were analysed by RT-PCR and normalized to HPRT mRNA levels. Data are mean ± SEM, n = 3–6, * p ≤ 0.05, *** p ≤ 0.001.

Figure 5

Respiratory chain complexes in complementation of ATP synthase deficiency. WB quantification in (A) liver and (B) heart of subunits of the Complex I (CI, NDUFA9, NDUFS3, NDUFB8), III (CIII, CORE2), and IV (CIV, COX4) related to Complex II (CII, SDHA) content was performed in control SHR rats and in SHR-Tmem70^ko/ko,tg/0 (tg/0) or SHR-Tmem70^ko/ko,tg/tg (tg/tg) rats derived from SHR-Tmem70^tg/tg transgenic lines 130 and 126. Data are mean ± SEM, n = 3–6, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 6

Full complementation of respiratory chain function in the liver. (A) Oxygen consumption in tissue homogenates was measured with CI (palmitoyl carnitine+pyruvate+glutamate) and CII (succinate) substrates in the presence of ADP (CI+CII coupled), oligomycin (leak), and FCCP (CI+CII maximal or CI maximal), RCR—respiratory control ratio. (B) Titration of sensitivity to oligomycin of ADP-stimulated respiration (CI+CII coupled) was analysed in control SHR rats and in SHR-Tmem70^ko/ko,tg/0 (tg/0) or SHR-Tmem70^ko/ko,tg/tg (tg/tg) derived from SHR-Tmem70^tg/tg transgenic lines 130 and 126. Data are mean ± SEM, n = 3–5.

Figure 7

Partial complementation of respiratory chain function in heart. (A) Oxygen consumption in tissue homogenates was measured with CI (palmitoyl carnitine+pyruvate+glutamate) and CII (succinate) substrates in the presence of ADP (CI+CII coupled), oligomycin (leak), and FCCP (CI+CII maximal or CI maximal), RCR—respiratory control ratio. (B) Titration of sensitivity to oligomycin of ADP-stimulated respiration (CI+CII coupled) was analysed in control SHR rats and in SHR-Tmem70^ko/ko,tg/0 (tg/0) or SHR-Tmem70^ko/ko,tg/tg (tg/tg) derived from SHR-Tmem70^tg/tg transgenic lines 130 and 126. Data are mean ± SEM, n = 3–6 * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 8

Systolic function of the left ventricle. The function of the hearts in control SHR rats and in SHR-Tmem70^ko/ko,tg/0 (tg/0) animals derived from SHR-Tmem70^tg/tg transgenic line 130 were analysed by echocardiography and were compared with the SHR controls. FS—fractional shortening, EF—ejection fraction. Data are mean ± SEM, n = 5–6, ** p ≤ 0.01.

Figure 9

Complementation of ATP synthase biogenesis in the heart. (A,C) Digitonin-solubilised heart proteins were resolved by blue native electrophoresis (BNE). ATP synthase monomers (cV_M), dimers (cV_D), and F₁ subcomplexes were detected by ATPase activity staining or the immunodetection of F₁-β or F_o-c subunits. Complex II content (assessed by SDHA antibody) was used for normalisation. Note that representative images in A for F₁-β and F_o-c originate from the same samples on the same membrane developed with different antibodies using two independent fluorescence channels. Therefore, only one SDHA reference is shown. (B,D) Quantification of the sum of all detected assemblies was performed in control SHR rats and in SHR-Tmem70^ko/ko,tg/0 (tg/0) or SHR-Tmem70^ko/ko,tg/tg (tg/tg) rats derived from SHR-Tmem70^tg/tg transgenic lines 130 and 126. Data are mean ± SEM, n = 3–4, ** p ≤ 0.01.

Figure 10

Respiratory chain Complex I biogenesis in the heart. (A,D) Digitonin-solubilized heart proteins were resolved by BNE electrophoresis and Complex I heterodimer with Complex III (I+III) and Complex I-containing supercomplexes (I+III+IV_n) were detected by the WB of the Ndufa9 or Ndufb8 subunits. (B,E) Heterodimer I+III content and (C,F) the sum of all Complex I assemblies (integrated signal from the area of the gel denoted by dark red line to the left of WB images) normalized to Complex II content (SDHA subunit) were quantified in control SHR rats and in two lines (130 and 126) of derived Tmem70^−/− knockout rats expressing the Tmem70 transgene at the single allele SHR-Tmem70^ko/ko,tg/0 (tg/0) or at both alleles SHR-Tmem70^ko/ko,tg/tg (tg/tg). Data are mean ± SEM, n = 5–13.