Blood mitochondrial DNA copy number: What are we counting?

Abstract

There is growing scientific interest to develop scalable biological measures that capture mitochondrial (dys)function. Mitochondria have their own genome, the mitochondrial DNA (mtDNA). It has been proposed that the number of mtDNA copies per cell (mtDNA copy number; mtDNAcn) reflects mitochondrial health. The common availability of stored DNA material or existing DNA sequencing data, especially from blood and other easy-to-collect samples, has made its quantification a popular approach in clinical and epidemiological studies. However, the interpretation of mtDNAcn is not univocal, and either a reduction or elevation in mtDNAcn can indicate dysfunction. The major determinants of blood-derived mtDNAcn are the heterogeneous cell type composition of leukocytes and platelet abundance, which can change with time of day, aging, and with disease. Hematopoiesis is a likely driver of blood mtDNAcn. Here we discuss the rationale and available methods to quantify mtDNAcn, the influence of blood cell type variations, and consider important gaps in knowledge that need to be resolved to maximize the scientific value around the investigation of blood mtDNAcn.

Keywords: Biomarker; Count; Leukocytes; Mitochondrial function; Mitochondrial genome; Mitochondrion; White blood cells.

Figure 1.. Mitochondrial DNA copy number (mtDNAcn) and relevant cellular contributors in human blood.

(A) Cell schematic with a nucleus (blue) and cytoplasmic mitochondria of various sizes and shapes. Most mitochondria contain one or more copies of mtDNA, all maternally inherited; each nucleus contains one pair of the nuclear genome, two copies or alleles, one from each parent. (B) The concept of mtDNA copy number (mtDNAcn), represented for different scenarios. Note that if all cells are identical, increasing the number of cells does not change the mtDNAcn. Platelets have mitochondria and mtDNA but do not have a nucleus, making it impossible to determine mtDNAcn in pure platelets (mtDNA copies divided by 0 = infinity). Contamination of white blood cells with platelets or cells with low mtDNAcn (e.g., granulocytes) can alter mtDNAcn. (C) Summary of mtDNAcn in different blood-derived cells. (D) Platelet count relative to all leukocytes in human blood (data from Rausser et al. 2021). (E) Summary of the direction and effect size of known influence of cell type composition for mtDNAcn in human blood, see text for details. (F) Micrograph of human saliva showing a similarly heterogenous cellular composition with several leukocytes (circled) and squamous buccal epithelial cells (arrows) that are shed from the inner lining of the cheeks.

Figure 2.. Hierarchy of biological processes linking mtDNA to mitochondrial health.

The mtDNA is the template for 13 mRNAs, which are translated into proteins in the mitochondrial matrix. All mtDNA-encoded proteins are subunits that assemble together with nDNA-encoded subunits into respiratory chain (RC) complexes within the inner mitochondrial membrane. RC complexes are subject to post-translational regulation that influence their ability to pump protons across the IMM and to respire (i.e., consume oxygen by complex IV). This generates the proton motive force that provide the driving force for ATP synthesis and other aspects of mitochondrial behavior. ATP synthesis by the OXPHOS system along with other dynamic processes of mitochondrial fusion/fission, signaling, and biogenesis, determine the integrated state of mitochondrial health that regulate cellular and organismal health. Thus, several independently modifiable biological steps separate the mtDNA template (mtDNAcn) from the final integrated function of mitochondria, explaining why in vivo mtDNAcn is not tightly coupled to energy production capacity nor with other physiologically-relevant aspect of mitochondrial health. Abbreviations: OXPHOS, oxidative phosphorylation; RC, respiratory chain.

Figure 3.. Summary of known factors contributing to mtDNAcn in human blood.

Preparations from left to right have increasing biological specificity, and consequently have more directly interpretable mtDNAcn. Whole blood is the complete (anti-coagulated) material collected, which contains all cellular constituents in variable proportions. Buffy coat is the complex cell mixture at the red blood cell-plasma interface after centrifugation of anti-coagulated blood. PBMCs, peripheral blood mononuclear cells, typically isolated using Ficoll 1077-based separation with centrifugation and platelet-depletion steps, which would deplete most granulocytes and a substantial portion of platelets. PBMCs can also be actively immuno-depleted of contaminating platelets using magnetic-activated cell sorting (MACS) with antibodies directed at platelet cell surface markers (e.g., CD41). Sorted cells are generated using a combination of positive and negative selection using MACS, or by flow cytometric cell sorting. Sorted cells represent the purest preparation with the highest level of biological specificity, and therefore allow the most directly interpretable measurement of cellular mtDNAcn.

Figure 4.. Theoretical scenarios for changes in mtDNAcn in purified cell type preparations or in human tissues.

(A) Both mtDNA depletion and excess mtDNA can indicate abnormal mitochondrial health, or mitochondrial dysfunction, as seen in rare cases of human disease (see text for details). mtDNAcn within specific cell subtypes exhibit substantial variation between individuals, and potentially even within individuals over time. (B) Potential etiology, mechanisms, and interpretation for reduced and (C) elevated mitochondrial mtDNAcn when measured in a purified cell population, after confounds related to cell composition have been ruled out. Abbreviations: dNTP, deoxynucleotide triphosphate; ROS, reactive oxygen species; 8-OHdG, 8-Oxo-2’-deoxyguanosine [a marker of oxidative DNA damage]; POLG, polymerase gamma [mtDNA polymerase]; TWINKLE, mtDNA helicase necessary for normal replication; RRM2B, ribonucleotide reductase regulatory TP53 inducible subunit M2B [a subunit of an enzyme involved in DNA synthesis]; TK2, thymidine kinase 2 [enzyme involved in DNA synthesis].