Metabolic shift underlies recovery in reversible infantile respiratory chain deficiency (2020)
Figure 3

Figure 3. Immunoblotting in skeletal muscle of patients and transcriptomics detected alterations of several mitochondrial complex subunits, the activation of integrated stress response and mTOR.

(A) Western blotting analysis depicts mitochondrial proteins (NDUFA9-Complex I, SDHA-Complex II, MTCO1-Complex IV and MTCO2-Complex IV) levels in control derived muscle (n=3), patient derived muscle during the symptomatic phase (n=3) and patient derived muscle after recovery (n=1). VCL was used as a loading control while the densitometry analysis was done based on the mitochondrial protein TOM20.

(B) Densitometry analysis of the immunoblotting from (A) showing an increase in SDHA (Complex II), while NDUFA9 (Complex I) probably as a compensatory mechanism and MT-CO2 (Complex IV) show significant reductions as the majority of mitochondrial encoded proteins belong to this two complexes, thus being destabilized by the defective mitochondrial mistranslation. Graphs show means ± s.d of triplicate samples (controls and affected muscle) and results obtained from (n=1) recovered muscle. For statistical analysis unpaired Student's t-test was employed where *p≤ 0.05 was considered statistically significant.

(C) Mitochondrial DNA copy numbers were determined in affected (n=3) and recovered individuals (n=2) and compared to control individuals (n=4, 3 months, 3 years, 4 years and 7 years) using qPCR showing a significant increase in recovered muscle. Graph shows mean ± s.d for each biological replicate. The average of controls are represented as horizontal line and graphs show mean ± s.d of replicates.

(D) Western blotting analysis of proteins involved in the amino-acid sensing and integrated stress response: GCN2, SLC7A11 and ATF4 in control derived muscle (n=3), patient derived muscle during the symptomatic phase (n=3) and patient derived muscle after recovery (n=1). All samples were normalized to VCL which was used as a loading control.

(E) Densitometry analysis of proteins from (D) showing a significant increase of major transducers of ISR such as GCN2 and ATF4. Consistent with ISR activation is also the increase in SLC7A1. Graphs show means ± s.d of triplicate samples (controls and affected muscle) and results obtained from n=1 recovered muscle. Similar results were observed in at least two independent results (due to the limited amount of sample). Statistical analysis unpaired Student's t-test was employed where *p≤ 0.05 was considered statistically significant.

(F) Transcriptomic pathways related to ATFs and ISR changed in affected patients compared to controls (RIRCD n=5; controls n=6) and in recovered muscle compared to affected muscle (n=1, affected and recovered muscle was collected from the same individual and was analysed using 4 technical replicates) and schematic representation of the metabolic pathways impacted in patient muscle (bottom, right panel). Results are expressed as fold change of the affected compared to controls or of the affected muscle compared to recovered and data are shown as mean ± SEM. For the calculation of p-value or padj we have employed the R package DESeq2 where the p-values obtained by the Wald test are corrected for multiple testing using the Benjamini and Hochberg method by default where *p ≤ 0.1.