The efficacy of treating acute myocardial ischemic damages depends, to a large extent, on the development of technologies for predicting their course and outcome. The aim of this paper was to explore whether it would be possible to consider the content of free circulating mitochondrial DNA as a danger-associated molecular pattern for assessing the probability of death from myocardial infarction. We have analyzed the clinical outcomes based on discharge summaries and autopsy reports obtained in the course of the PROTOCOL observational trial. This study was approved by the Irkutsk Scientific Center of Surgery and Traumatology ethics committee (protocol No. 3, 10.08.2015). To examine whether the assessment of the level of free circulating mtDNA in acute coronary syndrome can help predicting clinical outcomes, all patients were divided into two groups: group 1, involving those who survived during 30 days after hospitalization, and group 2, involving those who died during this time. A quantitative analysis of the free circulating mtDNA was conducted using the PCR method in situ. The analysis showed that in patients who survived the level of freely circulating mtDNA (36.0 copies/ml) was 164 times lower than in those who died (5900 copies/ml, p = 0.049). It should be mentioned that according to the logistic regression analysis, the probability of death of patients with the increased level of blood plasma mtDNA (more than 4000 copies/ml) is 50%. Thus, the PROTOCOL observational trial proved that the increase in the content of free circulating mtDNA in blood is a predictor of lethal outcome in patients with acute coronary syndrome. Trial registration The observational studies (those in which the assignment of the medical intervention is not at the discretion of the investigator) do not require registration.

Investigation and manipulation of mitochondrial genetics in animal and plant cells remains restricted by the lack of an efficient in vivo transformation methodology. Mitochondrial transfection in whole cells and maintenance of the transfected DNA are main issues on this track. We showed earlier that isolated mitochondria from different organisms can import DNA. Exploiting this mechanism, we assessed the possibility to maintain exogenous DNA in plant organelles. Whereas homologous recombination is scarce in the higher plant nuclear compartment, recombination between large repeats generates the multipartite structure of the plant mitochondrial genome. These processes are under strict surveillance to avoid extensive genomic rearrangements. Nevertheless, following transfection of isolated organelles with constructs composed of a partial gfp gene flanked by fragments of mitochondrial DNA, we demonstrated in organello homologous recombination of the imported DNA with the resident DNA and integration of the reporter gene. Recombination yielded insertion of a continuous exogenous DNA fragment including the gfp sequence and at least 0.5 kb of flanking sequence on each side. According to our observations, transfection constructs carrying multiple sequences homologous to the mitochondrial DNA should be suitable and targeting of most regions in the organelle genome should be feasible, making the approach of general interest.

Mitochondrial gene products are essential for the viability of eukaryote obligate aerobes. Consequently, mutations of the mitochondrial genome cause severe diseases in man and generate traits widely used in plant breeding. Pathogenic mutations can often be identified but direct genetic rescue remains impossible because mitochondrial transformation is still to be achieved in higher eukaryotes. Along this line, it has been shown that isolated plant and mammalian mitochondria are naturally competent for importing linear DNA. However, it has proven difficult to understand how such large polyanions cross the mitochondrial membranes. The genetic tractability of Saccharomyces cerevisae could be a powerful tool to unravel this molecular mechanism. Here we show that isolated S. cerevisiae mitochondria can import linear DNA in a process sharing similar characteristics to plant and mammalian mitochondria. Based on biochemical data, translocation through the outer membrane is believed to be mediated by voltage-dependent anion channel (VDAC) isoforms in higher eukaryotes. Both confirming this hypothesis and validating the yeast model, we illustrate that mitochondria from S. cerevisiae strains deleted for the VDAC-1 or VDAC-2 gene are severely compromised in DNA import. The prospect is now open to screen further mutant yeast strains to identify the elusive inner membrane DNA transporter.

In a number of dicotyledonous plants, including Arabidopsis, the transcription of organellar genes is performed by three nuclear-encoded RNA polymerases, RPOTm, RPOTmp, and RPOTp. RPOTmp is a protein with a dual targeting, which is presumably involved in the control of gene expression in both mitochondria and chloroplasts. A previous study of the Arabidopsis insertion rpotmp mutant showed that it has retarded growth and development, altered leaf morphology, changed expression of mitochondrial and probably some chloroplast genes, and decreased activities of the mitochondrial respiratory complexes. To date, there is no clear evidence as to which of these disorders are associated with a lack of RPOTmp in each of the two organelles. The aim of this study was to elucidate the role that this RNA polymerase specifically plays in mitochondria and chloroplasts. Two sets of Arabidopsis transgenic lines with complementation of RPOTmp function in either mitochondria or chloroplasts were obtained. It was found that the recovery of RPOTmp RNA polymerase activity in chloroplasts, although restoring the transcription from the RPOTmp-specific PC promoter, did not lead to compensation of the mutant growth defects. In contrast, the rpotmp plants expressing RPOTmp with mitochondrial targeting restored the level of mitochondrial transcripts and exhibit a phenotype resembling that of the wild-type plants. We conclude that despite its localization in two cell compartments, Arabidopsis RPOTmp plays an important role in mitochondria, but not in chloroplasts.