Assessment of Mitochondrial Metabolic Oxidative State in Living Cardiomyocytes with Spectrally-Resolved Fluorescence Lifetime Spectroscopy of NAD(P)H
Bibliographic record
Abstract
The primary function of cardiac mitochondria is the production of ATP to support heart contraction. Examination of the mitochondrial redox state is therefore crucially important to sensitively detect early signs of mitochondrial function in pathophysiological conditions,such as ischemia,diabetes and heart failure. We study fingerprinting of mitochondrial metabolic oxidative state in living cardiomyocytes with spectrally-resolved fluorescence lifetime spectroscopy of NAD(P)H,the principal electron donor in mitochondrial respiration responsible for vital ATP supply. Here NAD(P)H is studied as a marker for non-invasive fluorescent probing of the mitochondrial function. NAD(P)H fluorescence is recorded in cardiac cells following excitation with 375nm UV-light and detection by spectrally-resolved time-correlated single photon counting (TCSPC),based on the simultaneous measurement of the fluo-rescence spectra and fluorescence lifetimes. Modulation of NADH production and/or mitochondrial respiration is tested to study dynamic characteristics of NAD(P)H fluorescence decay. Our results show that at least a 3-exponential decay model,with 0.4-0.7ns,1.2-1.9ns and 8.0-13.0ns lifetime pools is necessary to describe cardiomyocyte autofluorescence (AF) within 420-560nm spectral range. Increased mitochondrial NADH production by ketone bodies enhanced the fluorescence intensity,without significant change in fluorescent lifetimes. Rotenone,the inhibitor of Complex I of the mitochondrial respiratory chain,increased AF intensity and shortened the average fluorescence lifetime. Dinitrophenol (DNP),an uncoupling agent of the mitochondrial oxidative phosphorylation,lowered AF intensity,broadened the spectral shoulder at 520 nm and increased the average fluorescence lifetime. These effects are comparable to the study of NADH fluorescence decay in vitro. In the present contribution we demonstrated that spectrally-resolved fluorescence lifetime technique provides promising new tool for analysis of mitochondrial NAD(P)H fluorescence with good reproducibility in living cardiomyocytes. This approach will enhance our knowledge about cardiomyocyte oxidative metabolism and /or its dysfunction at a cellular level. In the future,this approach can prove helpful in the clinical diagnosis and treatment of mitochondrial disorder.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| 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) | 0.001 | 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 source (direct Gemma or distilled Codex), 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".