Cells expressing POLG loss-of-function variants can decrease mtDNA copy number and increased growth rates in neighbouring A7r5 and HeLa cells
Bibliographic record
Abstract
Mitochondrial disfunction promotes vascular disease, and mitochondrial diseases offer valuable insights into the pathological mechanisms of mitochondrial dysfunction. We previously described a patient cohort with variants in the mitochondrial DNA gamma polymerase (POLG) that were prone hypertension. We hypothesized that the POLG variants promote hypertrophy or hyperplasia of vascular smooth muscle cells in a way that stiffens or thickens blood vessels to explain the high rate of resistant hypertension in this cohort. The objective of this study was to assess the cellular phenotype of cells over-expressing POLG variants: pathogenic Y955C and variants of unknown significance; R964C, I1098N, Y1138C. We transiently expressed human FLAG-tagged POLG variants in rat A7r5 and HeLa cells for 2-4 days. Transfection effciency ranged from 20-30% in A7r5 and >60% in HeLa cells. POLG variants increased cell density by nuclear counting (20-60%) and growth rates measured by 48-72 h of live-cell imaging (Y955C > R964C > I1089N » Y1138C). Compared to wild-type, Y955C and R964C reduced mtDNA copy number (mCN) measured by droplet digital PCR by 10-20%. Imaging mtDNA nucleoids (anti-dsDNA) and the FLAG-tag showed that reduced mCN was not restricted to transfected cells. Addition of a P2A-T2A-nuclear-EGFP to the POLG constructs demonstrated that faster growth rates were not specific to transfected cells. Resting and oligomycin-stimulated mitochondrial membrane potential imaged with TMRM were unaffected by Y955C and R964C. Mitochondrial ROS (mitoSox) was similar between cultures transfected with Wild-type versus POLG variants and lower than levels stimulated by antimycin-A’s inhibition of complex III. Notably, we found a linear relationship between mCN and cell density that was not simply due to smaller cell size at higher densities. Using Hoescht-33342 staining to assess cell cycle, no clear difference in cell-cycle distribution was evident in unsynchronized cultures. We conclude that modest reduction of mCN induces a mitogenic effect, likely via a diffusible messenger that further reduces mCN in a cell population. The signalling mechanism of this phenomenon remains to be identified, but our results confirm that POLG malfunction can alter smooth muscle physiology in a way that could support pathogenic vascular remodelling and could promote enhanced growth in cancerous cells. Funded by the Natural Sciences and Research Council of Canada. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".