The alpha-ketoglutarate dehydrogenase complex (KGDHc), also known as the 2-oxoglutarate dehydrogenase complex, plays a crucial role in oxidative metabolism. It catalyzes a key step in the tricarboxylic acid (TCA) cycle, producing NADH (primarily for oxidative phosphorylation) and succinyl-CoA (for substrate-level phosphorylation, among others). Additionally, KGDHc is also capable of generating reactive oxygen species, which contribute to mitochondrial oxidative stress. Hence, the KGDHc and its dysfunction are implicated in various pathological conditions, including selected neurodegenerative diseases. The pathological roles of KGDHc in these diseases are generally still obscure. The aim of this study was to assess whether the mitochondrial malfunctions observed in the dihydrolipoamide succinyltransferase (DLST) and dihydrolipoamide dehydrogenase (DLD) double-heterozygous knockout (DLST+/-DLD+/-, DKO) mice are associated with neuronal and/or metabolic abnormalities. In the DKO animals, the mitochondrial O2 consumption and ATP production rates both decreased in a substrate-specific manner. Reduced H2O2 production was also observed, either due to Complex I inhibition with α-ketoglutarate or reverse electron transfer with succinate, which is significant in ischaemia-reperfusion injury. Middle-aged DKO mice exhibited minor cognitive decline, associated with microgliosis in the cerebral cortex and neuronal death in the Cornu Ammonis subfield 1 (CA1) of the hippocampus, indicating neuroinflammation. This was supported by increased levels of dynamin-related protein 1 (Drp1) and reduced levels of mitofusin 2 and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in DKO mice. Observations on activity, food and oxygen consumption, and blood amino acid and acylcarnitine profiles revealed no significant differences. However, middle-aged DKO animals showed decreased performance in the treadmill fatigue-endurance test as compared to wild-type animals, accompanied by subtle resting cardiac impairment, but not skeletal muscle fibrosis. In conclusion, DKO animals compensate well the double-heterozygous knockout condition at the whole-body level with no major phenotypic changes under resting physiological conditions. However, under high energy demand, middle-aged DKO mice exhibited reduced performance, suggesting a decline in metabolic compensation. Additionally, microgliosis, neuronal death, decreased mitochondrial biogenesis, and altered mitochondrial dynamics were observed in DKO animals, resulting in minor cognitive decline. This is the first study to highlight the in vivo changes of this combined genetic modification. It demonstrates that unlike single knockout rodents, double knockout mice exhibit phenotypical alterations that worsen under stress situations.

Defect of GTPBP3, the human mitochondrial tRNA-modifying enzyme, can lead to Combined Oxidative Phosphorylation Deficiency 23 (COXPD23). Up to now, about 20 different variants of the GTPBP3 gene have been reported; however, genotype-phenotype analysis has rarely been described. Here, we reported a 9-year-old boy with COXPD23 who presented with hyperlactatemia, hypertrophic cardiomyopathy, seizures, feeding difficulties, intellectual disability and motor developmental delay, and abnormal visual development. Biallelic pathogenic variants of the GTPBP3 gene were identified in this boy, one novel variant c.1102dupC (p. Arg368Profs*22) inherited from the mother and the other known variant c.689A>C (p. Gln230Pro) inherited from father. We curated 18 COXPD23 patients with GTPBP3 variants to investigate the genotype-phenotype correlation. We found that hyperlactatemia and cardiomyopathy were critical clinical features in COXPD23 and the average onset age was 1.7 years (3 months of age for the homozygote). Clinical classification of COXPD23 for the two types, severe and mild, was well described in this study. We observed arrhythmia and congestive heart failure frequently in the severe type with early childhood mortality, while developmental delay was mainly observed in the mild type. The proportion of homozygous variants (71.4%) significantly differed from that of compound heterozygous variants (18.1%) in the severe type. Compared with the variants in gnomAD, the proportion of LOFVs in GTPBP3 was higher in COXPD23 patients (48.6% versus 8.9%, p < 0.0001 ****), and 31% of them were frameshift variants, showing the LOF mechanism of GTPBP3. Additionally, the variants in patients were significantly enriched in the TrmE-type G domain, indicating that the G domain was crucial for GTPBP3 protein function. The TrmE-type G domain contained several significant motifs involved in the binding of guanine nucleotides and Mg2+, the hydrolysis of GTP, and the regulation of the functional status of GTPases. In conclusion, we reported a mild COXPD23 case with typical GTPBP3-related symptoms, including seizures and abnormal visual development seldom observed previously. Our study provides novel insight into understanding the clinical diagnosis and genetic counseling of patients with COXPD23 by exploring the genetic pathogenesis and genotype-phenotype correlation of COXPD23.

Cardiovascular complications are the major cause of mortality and morbidity in diabetic patients. The changes in myocardial structure and function associated with diabetes are collectively called diabetic cardiomyopathy. Numerous molecular mechanisms have been proposed that could contribute to the development of diabetic cardiomyopathy and have been studied in various animal models of type 1 or type 2 diabetes. The current review focuses on the role of sodium (Na+) in diabetic cardiomyopathy and provides unique data on the linkage between Na+ flux and energy metabolism, studied with non-invasive 23Na, and 31P-NMR spectroscopy, polarography, and mass spectroscopy. 23Na NMR studies allow determination of the intracellular and extracellular Na+ pools by splitting the total Na+ peak into two resonances after the addition of a shift reagent to the perfusate. Using this technology, we found that intracellular Na+ is approximately two times higher in diabetic cardiomyocytes than in control possibly due to combined changes in the activity of Na+-K+ pump, Na+/H+ exchanger 1 (NHE1) and Na+-glucose cotransporter. We hypothesized that the increase in Na+ activates the mitochondrial membrane Na+/Ca2+ exchanger, which leads to a loss of intramitochondrial Ca2+, with a subsequent alteration in mitochondrial bioenergetics and function. Using isolated mitochondria, we showed that the addition of Na+ (1-10 mM) led to a dose-dependent decrease in oxidative phosphorylation and that this effect was reversed by providing extramitochondrial Ca2+ or by inhibiting the mitochondrial Na+/Ca2+ exchanger with diltiazem. Similar experiments with 31P-NMR in isolated superfused mitochondria embedded in agarose beads showed that Na+ (3-30 mM) led to significantly decreased ATP levels and that this effect was stronger in diabetic rats. These data suggest that in diabetic cardiomyocytes, increased Na+ leads to abnormalities in oxidative phosphorylation and a subsequent decrease in ATP levels. In support of these data, using 31P-NMR, we showed that the baseline β-ATP and phosphocreatine (PCr) were lower in diabetic cardiomyocytes than in control, suggesting that diabetic cardiomyocytes have depressed bioenergetic function. Thus, both altered intracellular Na+ levels and bioenergetics and their interactions may significantly contribute to the pathology of diabetic cardiomyopathy.

RMND1 is an integral inner membrane mitochondrial protein that assembles into a large 240 kDa complex to support translation of the 13 polypeptides encoded on mtDNA, all of which are essential subunits of the oxidative phosphorylation (OXPHOS) complexes. Variants in RMND1 produce global defects in mitochondrial translation and were first reported in patients with severe neurological phenotypes leading to mortality in the first months of life. Using whole-exome sequencing, we identified compound heterozygous RMND1 variants in a 4-year-old patient with congenital lactic acidosis, severe myopathy, hearing loss, renal failure, and dysautonomia. The levels of mitochondrial ribosome proteins were reduced in patient fibroblasts, causing a translation defect, which was rescued by expression of the wild-type cDNA. RMND1 was almost undetectable by immunoblot analysis in patient muscle and fibroblasts. BN-PAGE analysis showed a severe combined OXPHOS assembly defect that was more prominent in patient muscle than in fibroblasts. Immunofluorescence experiments showed that RMND1 localizes to discrete foci in the mitochondrial network, juxtaposed to RNA granules where the primary mitochondrial transcripts are processed. RMND1 foci were not detected in patient fibroblasts. We hypothesize that RMND1 acts to anchor or stabilize the mitochondrial ribosome near the sites where the mRNAs are matured, spatially coupling post-transcriptional handling mRNAs with their translation, and that loss of function variants in RMND1 are associated with a unique constellation of clinical phenotypes that vary with the severity of the mitochondrial translation defect.