Background: Evidence supports an important link between mitochondrial DNA (mtDNA) variation and adverse drug reactions such as idiosyncratic drug-induced liver injury (iDILI). Here, we describe the generation of HepG2-derived transmitochondrial cybrids, to investigate the impact of mtDNA variation on mitochondrial (dys)function and susceptibility to iDILI. This study created 10 cybrid cell lines, each containing distinct mitochondrial genotypes of haplogroup H or haplogroup J backgrounds.
Methods: HepG2 cells were depleted of mtDNA to make rho zero cells, before the introduction of known mitochondrial genotypes using platelets from healthy volunteers (n=10), thus generating 10 transmitochondrial cybrid cell lines. The mitochondrial function of each was assessed at basal state and following treatment with compounds associated with iDILI; flutamide, 2-hydroxyflutamide, and tolcapone, and their less toxic counterparts bicalutamide and entacapone utilising ATP assays and extracellular flux analysis.
Findings: Whilst only slight variations in basal mitochondrial function were observed between haplogroups H and J, haplogroup-specific responses were observed to the mitotoxic drugs. Haplogroup J showed increased susceptibility to inhibition by flutamide, 2-hydroxyflutamide and tolcapone, via effects on selected mitochondrial complexes (I and II), and an uncoupling of the respiratory chain.
Conclusions: This study demonstrates that HepG2 transmitochondrial cybrids can be created to contain the mitochondrial genotype of any individual of interest. This provides a practical and reproducible system to investigate the cellular consequences of variation in the mitochondrial genome, against a constant nuclear background. Additionally, the results show that inter-individual variation in mitochondrial haplogroup may be a factor in determining sensitivity to mitochondrial toxicants.
Funding: This work was supported by the Centre for Drug Safety Science supported by the Medical Research Council, United Kingdom (Grant Number G0700654); and GlaxoSmithKline as part of an MRC-CASE studentship (grant number MR/L006758/1).
Source Data files have been provided for Figures 2, 3, 4, 5, 6 and supplementary figures s1, s3, s4, s5, s6
- Carol Elizabeth Jolly
- Amy E Chadwick
- Amy Louise Ball
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Human subjects: This work with human material in this project was approved by the North West of England Research Ethics Committee and all participants gave written informed consent and consent to publish. All procedures were in accordance with the ethical standards of the North West of England Research Ethics Committee (Cell Archive of HLA Typed Healthy Volunteers (HLA), CRN ID 7787, IRAS ID: 15623) with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
- Ilse S Daehn, Icahn School of Medicine at Mount Sinai, United States
© 2023, Ball et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
To systematically identify cell types in the human ligament, investigate how ligamental cell identities, functions, and interactions participated in the process of ligamental degeneration, and explore the changes of ligamental microenvironment homeostasis in the disease progression.
Using single-cell RNA sequencing and spatial RNA sequencing of approximately 49,356 cells, we created a comprehensive cell atlas of healthy and degenerated human anterior cruciate ligaments. We explored the variations of the cell subtypes’ spatial distributions and the different processes involved in the disease progression, linked them with the ligamental degeneration process using computational analysis, and verified findings with immunohistochemical and immunofluorescent staining.
We identified new fibroblast subgroups that contributed to the disease, mapped out their spatial distribution in the tissue and revealed two dynamic trajectories in the process of the degenerative process. We compared the cellular interactions between different tissue states and identified important signaling pathways that may contribute to the disease.
This cell atlas provides the molecular foundation for investigating how ligamental cell identities, biochemical functions, and interactions contributed to the ligamental degeneration process. The discoveries revealed the pathogenesis of ligamental degeneration at the single-cell and spatial level, which is characterized by extracellular matrix remodeling. Our results provide new insights into the control of ligamental degeneration and potential clues to developing novel diagnostic and therapeutic strategies.
This study was funded by the National Natural Science Foundation of China (81972123, 82172508, 82372490) and 1.3.5 Project for Disciplines of Excellence of West China Hospital Sichuan University (ZYJC21030, ZY2017301).
Low-protein (LP) diets extend the lifespan of diverse species and are associated with improved metabolic health in both rodents and humans. Paradoxically, many athletes and bodybuilders consume high-protein (HP) diets and protein supplements, yet are both fit and metabolically healthy. Here, we examine this paradox using weight pulling, a validated progressive resistance exercise training regimen, in mice fed either an LP diet or an isocaloric HP diet. We find that despite having lower food consumption than the LP group, HP-fed mice gain significantly more fat mass than LP-fed mice when not exercising, while weight pulling protected HP-fed mice from this excess fat accretion. The HP diet augmented exercise-induced hypertrophy of the forearm flexor complex, and weight pulling ability increased more rapidly in the exercised HP-fed mice. Surprisingly, exercise did not protect from HP-induced changes in glycemic control. Our results confirm that HP diets can augment muscle hypertrophy and accelerate strength gain induced by resistance exercise without negative effects on fat mass, and also demonstrate that LP diets may be advantageous in the sedentary. Our results highlight the need to consider both dietary composition and activity, not simply calories, when taking a precision nutrition approach to health.