Mitochondrial DNA haplogroups modify the risk of osteoarthritis by altering mitochondrial function and intracellular mitochondrial signals

Abstract

Haplogroup G predisposes one to an increased risk of osteoarthritis (OA) occurrence, while haplogroup B4 is a protective factor against OA onset. However, the underlying mechanism is not known. Here, by using trans-mitochondrial technology, we demonstrate that the activity levels of mitochondrial respiratory chain complex I and III are higher in G cybrids than in haplogroup B4. Increased mitochondrial oxidative phosphorylation (OXPHOS) promotes mitochondrial-related ATP generation in G cybrids, thereby shifting the ATP generation from glycolysis to OXPHOS. Furthermore, we found that lower glycolysis in G cybrids decreased cell viability under hypoxia (1% O2) compared with B4 cybrids. In contrast, G cybrids have a lower NAD(+)/NADH ratio and less generation of reactive oxygen species (ROS) under both hypoxic (1% O2) and normoxic (20% O2) conditions than B4 cybrids, indicating that mitochondrial-mediated signaling pathways (retrograde signaling) differ between these cybrids. Gene expression profiling of G and B4 cybrids using next-generation sequencing technology showed that 404 of 575 differentially expressed genes (DEGs) between G and B4 cybrids are enriched in 17 pathways, of which 11 pathways participate in OA. Quantitative reverse transcription PCR (qRT-PCR) analyses confirmed that G cybrids had lower glycolysis activity than B4 cybrids. In addition, we confirmed that the rheumatoid arthritis pathway was over-activated in G cybrids, although the remaining 9 pathways were not further tested by qRT-PCR. In conclusion, our findings indicate that mtDNA haplogroup G may increase the risk of OA by shifting the metabolic profile from glycolysis to OXPHOS and by over-activating OA-related signaling pathways.

Generation and Bioenergetic Profiles of Cybrids with East Asian mtDNA Haplogroups

“…Cybrids were formed by fusing 143B ρ 0 cells and platelets from healthy individuals with different haplotypes [15]. Cybrid clones were cultured in high-glucose DMEM containing 10% fetal bovine serum at 37°C in an atmosphere with 5% carbon dioxide.…”

“…Genomic DNA from the volunteers was extracted using sodium dodecyl sulfate (SDS) lysis buffer as described previously [8]. Sanger sequencing was performed for the entire mtDNA of each individual on an ABI 3730XL system (Thermo Fisher Scientific) with 24 primer pairs [15]. Detailed mtDNA haplotypes were annotated for each subject based on an established mtDNA tree [11, 12].…”

“…The NAD + /NADH ratio was detected in cultured cells with an NAD + /NADH ratio assay kit (Abcam, Cambridge, MA, USA) [15]. Briefly, cells were washed with phosphate-buffered saline (PBS) buffer and lysed with lysis buffer for 15 minutes at room temperature.…”

“…A phylogenetic tree of L3 subhaplogroups that migrated from Africa to East Asia [11, 12] revealed that many of the subclades were associated with metabolic and degenerative diseases [13, 14]. The link between mtDNA haplogroup/SNPs and disease has been attributed to possible changes in mitochondrial reactive oxygen species (ROS) levels, respiration capacity, and ATP production [15, 16]. Functional analysis of mtDNA SNPs identified a common mtDNA variation, m.9821insA/AA, that affected mitochondrial ROS generation and OxPhos in mouse cells [17], while another showed that mtDNA haplogroups influence phenotypic variability in mice [18].…”

“…Haplogroups related or unrelated to disease were fused with human 143B osteosarcoma cells lacking mtDNA rho zero ( ρ 0) cells [19], respectively. The effects of East Asian haplogroups/SNPs G versus B4 on osteoarthritis [15] and m.8584A/10398G versus m.8584G/10398A on Parkinson's disease [20] and of haplogroup B4 versus D4 on diabetes [14] and haplogroup B4 and E on biliary atresia [21] have been analyzed. However, mitochondrial function profile of the entire mtDNA phylogenetic tree was not reported.…”