The proximal domains of mitochondria and the endoplasmic reticulum (ER) are linked by tethering factors on each membrane, allowing the efficient transport of substances, including lipids and calcium, between them. However, little is known about the regulation and function of mitochondria-ER contacts (MERCs) dynamics under mitochondrial damage. In this study, we apply NanoBiT technology to develop the MERBiT system, which enables the measurement of reversible MERCs formation in living cells. Analysis using this system suggests that induction of mitochondrial ROS increases MERCs formation via RMDN3 (also known as PTPIP51)-VAPB tethering driven by RMDN3 phosphorylation. Disruption of this tethering caused lipid radical accumulation in mitochondria, leading to cell death. The lipid radical transfer activity of the TPR domain in RMDN3, as revealed by an in vitro liposome assay, suggests that RMDN3 transfers lipid radicals from mitochondria to the ER. Our findings suggest a potential role for MERCs in cell survival strategy by facilitating the removal of mitochondrial lipid radicals under mitochondrial damage.

Mitochondrial respiratory chain complexes II, III, and IV and cytochrome c contain haem, which is generated by the insertion of Fe²⁺ into protoporphyrin IX. 5-Aminolevulinic acid (ALA) combined with sodium ferrous citrate (SFC) was reported to enhance haem production, leading to respiratory complex and haem oxygenase-1 (HO-1) upregulation. Here, we investigated the effects of different concentrations of ALA and SFC alone or in combination (ALA/SFC) on fibroblasts from 8 individuals with mitochondrial diseases and healthy controls. In normal fibroblasts, expression levels of oxidative phosphorylation (OXPHOS) complex subunits and corresponding genes were upregulated only by ALA/SFC. Additionally, the increased oxygen consumption rate (OCR) and ATP levels in normal fibroblasts were more obvious after treatment with ALA/SFC than after treatment with ALA or SFC. OXPHOS complex proteins were enhanced by ALA/SFC, whereas OCR and ATP levels were increased in 6 of the 8 patient-derived fibroblasts. Further, HO-1 protein and mRNA levels were enhanced by ALA/SFC in all fibroblasts. The relative mtDNA copy number was increased by ALA/SFC. Thus, our findings indicate that ALA/SFC is effective in elevating OXPHOS, HO-1 protein, and mtDNA copy number, resulting in an increase in OCR and ATP levels, which represents a promising therapeutic option for mitochondrial diseases.

Cardiomyopathy is a risk factor for poor prognosis in pediatric patients with mitochondrial disease. However, other risk factors including genetic factors related to poor prognosis in mitochondrial disease has yet to be fully elucidated. Between January 2004 and September 2019, we enrolled 223 consecutive pediatric mitochondrial disease patients aged <18 years with a confirmed genetic diagnosis, including 114 with nuclear gene mutations, 89 patients with mitochondrial DNA (mtDNA) point mutations, 11 with mtDNA single large-scale deletions and 9 with chromosomal aberrations. Cardiomyopathy at baseline was observed in 46 patients (21%). Hazard ratios (HR) and 95% confidence intervals (CI) were calculated for all-cause mortality. Over a median follow-up of 36 months (12-77), there were 85 deaths (38%). The overall survival rate was significantly lower in patients with cardiomyopathy than in those without (p < 0.001, log-rank test). By multivariable analysis, left ventricular (LV) hypertrophy (HR = 4.6; 95% CI: 2.8-7.3), neonatal onset (HR = 2.9; 95% CI: 1.8-4.5) and chromosomal aberrations (HR = 2.9; 95% CI: 1.3-6.5) were independent predictors of all-cause mortality. Patients with LV hypertrophy with neonatal onset and/or chromosomal aberrations had higher mortality (100% in 21 patients) than those with LV hypertrophy alone (71% in 14 patients). In pediatric patients with mitochondrial disease, cardiomyopathy was common (21%) and was associated with increased mortality. LV hypertrophy, neonatal onset and chromosomal aberrations were independent predictors of all-cause mortality. Prognosis is particularly unfavorable if LV hypertrophy is combined with neonatal onset and/or chromosomal aberrations.