Different tissues have different energy requirements. These energy requirements can affect mtDNA copy number as well as the relative amounts of exposure to ROS originating from the respiratory complexes. Both of these variables might impose requirements for mtDNA maintenance.
Furthermore, the molecular mechanisms required for the physiologically-meaningful mtDNA maintenance might be affected in various pathological conditions. In order to gather supporting information of the possible involvement of these variables in modulating of mtDNA replication and/or organization, I measured copy number in different human tissues. I also analyzed the effects of deliberately provoking oxidative stress to mtDNA and examined post mortem heart mtDNA replication and recombination intermediates from a person diagnosed with a severe ischemic heart disease. All this data, whilst provocative is very preliminary and more experiments are needed. I would like to present these results only as perspectives for future work.
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5.3.1 mtDNA copy number in different tissues (IV)
The mtDNA content of the cell depends on its energy requirements (Moraes 2001). Because mtDNA must be packaged into nucleoids, the copy number must be related either to the size or number of nucleoids. However, nothing is known about how mtDNA is organized in nucleoids. It is plausible that the high MW mtDNA complexes found in human heart (Chapter 5.1) represent the nucleoid organization of mtDNA in these cells. With more mtDNA molecules per nucleoid, the opportunity for inter-molecular recombination would increase, providing an explanation for the high levels of recombination seen in human heart as well as possible resistance to ischemic stress.
In order to see whether mtDNA organization correlated with copy number in different tissues, I measured mtDNA copy number using real time quantitative PCR (qPCR) by comparing the relative quantity of mtDNA to that of a typical diploid nuclear gene. Heart had the highest copy number of around 11,000 and lymph node the lowest of around 500 mtDNA copies per cell.
Skeletal muscle was estimated to have 2300 copies and all other tissues around 5000 copies of mtDNA per cell (Figure 5.28).
Figure 5.28. mtDNA copy number per diploid nuclear gene in various human tissues from one individual as measured by qPCR.
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5.3.2 Oxidative damage of mtDNA and replication stall (unpublished data)
Heart is a continuously-working tissue and it is expected that the highly-active OXPHOS system also produces comparatively high amounts of ROS. ROS are a known cause of DNA damage, including double-strand breaks (DSBs [David et al. 2007]). DSBs can result in genomic rearrangements or possibly in a complete loss of a chromosome. As recombination-mediated DNA repair is the most efficient means of repairing DSBs, a highly active molecular recombination system in heart might be a physiologically-regulated response to the fact that heart mtDNA is constantly bombarded by ROS. To evaluate whether recombination or other effects on mtDNA replication could be produced in cell culture by oxidative damage, HEK293T cells were exposed to various amounts of KBrO3, which is known to be a potent oxidizer of DNA in vivo. The oxidative damage in mtDNA was confirmed by measuring the relative amount of nicks per mtDNA molecule using alkaline AGE after treating the sample with E. coli Fpg, which is an 8-oxo-deoxyguanosine (8-OHdG) DNA glycosylase. Eight hours of exposure to 30 mM KBrO3 induced changes in the pattern of mtDNA RIs as seen by 2DNAGE, and 24h exposure resulted in the accumulation of replication intermediates indicating severe stalling of replication (Figure 5.29). However, X-arcs were not influenced by this treatment.
Figure 5.29. Oxidative damage by potassium bromate on HEK293T cells after 24h exposure. BclI digest, ND2 probe. Note increase in the standard Y-arc (arrow) resulting from accumulating replication intermediates, indicative of replication stall. RITOLS intermediates are not significantly affected. No increase can be observed in the recombination intermediates.
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5.3.3 Modified mtDNA RIs in a case of a ischemic heart disease
(unpublished data)
There are indications that certain cardiac pathologies either directly or indirectly affect mtDNA copy number or other functions in the diseased heart. However, a survey of somatic mtDNA rearrangements and heart pathology did not reveal any causal relationship with common heart diseases (Kajander et al. 2002). The mtDNA rearrangements in heart rather seem to have a loose age-dependent equilibrium with normal mtDNA, indicating that they arise from a non-pathological process. Despite being only a small sample, the heart pathologies checked in the study revealed some interesting outliers. Instead of having elevated numbers of rearrangements as one would expect, some cases had almost undetectable amounts of them. If the rearrangements result from an active recombination mechanism, their absence might indicate a switch to a more conventional DNA replication, which could itself be cited to pathology, if recombination-dependent processes are protective against mtDNA damage. This notion is supported by 2DNAGE, which I carried out on mtDNA from an individual with severe ischemic heart disease. In this case as a distinction from all other individuals that I surveyed, strong RITOLS intermediates can be detected and there are much less X-forms than in control heart samples (Figure 5.26.). This observation is thus far anecdotal, but raises interesting future perspectives (see Discussion).
Figure 5.26. Unusual PvuII and DraI 2DNAGE patterns of heart mtDNA from a person who suffered from severe ischemic heart muscle disease during his lifetime. Compare with Figure 5.7 above and Figure 2b of original communication IV. Strong theta- and RITOLS intermediates can be detected.
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6 DISCUSSION
For nearly the last three decades it was thought that the mechanism of mammalian mitochondrial DNA replication had been elucidated thoroughly and only the identification of the relevant replicative proteins and their characterization remained. While the investigation of plant and yeast mtDNA maintenance has been hampered by the evident complexity of the processes involved, much of the mammalian mtDNA research has been biased by the assumptions of mechanistical simplicity and failure to examine tissue- or species-specific features. Most of the research has been concentrated on cultured cells or on only one model mammal – the mouse. The work by Kajander et al. (2001) was one of the first indications that the extrapolated view is inaccurrate, because it described for the first time abundant recombination as a completely new feature of mammalian mtDNA. The observation was obviously not just anecdotal, as the relative quantities of junctional molecules were much higher than those reported in other systems with well-documented active recombination. It was obvious that these molecules represented some central aspects of mtDNA maintenance in healthy human heart, although the finding has been largely ignored by the field.
In this series of studies, I conducted extensive investigations of mammalian mtDNA replication under various conditions in cell culture as well in different tissues of the organism. I found new functions for genes known to be involved in mitochondrial transcription or mtDNA maintenance, characterized replication phenotypes of catalytically-defective replisome proteins, revealed physiologically-significant tissue-specific differences in mtDNA replication, and showed that these features could be manipulated in a transgenic model organism. Finally, by applying several different analytical methods in concert, I revealed evidence for a novel mechanism of mtDNA replication in human heart. Taken together, the work published in original communications I-IV has helped to deepen our understanding of the maintenance of mammalian mtDNA.