Real-Time Assessment of Mitochondrial ATP Synthesis Response: MitoRaise

Academic Background

Mitochondria are the energy factories within cells, primarily synthesizing adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS). ATP serves as the main energy carrier in cells, and its synthesis rate directly reflects the functional status of mitochondria. However, traditional ATP measurement methods typically rely on single-point measurements of ATP levels or indirect assessments of mitochondrial function through oxygen consumption rates (OCR), which cannot monitor the dynamic changes in ATP synthesis in real time. Particularly in diseases such as cancer, abnormalities in mitochondrial metabolism are closely related to disease progression, making it crucial to develop a method capable of real-time monitoring of mitochondrial ATP synthesis rates.

To address this issue, a research team from Sungkyunkwan University and Samsung Medical Center developed a new technology called MitoRaise, aimed at measuring mitochondrial ATP synthesis rates in real time and exploring its potential applications in disease monitoring.

Source of the Paper

The study was conducted by Eun Sol Chang, Kyoung Song, Ji-Young Song, and other authors from multiple institutions, including Sungkyunkwan University and Samsung Medical Center. The paper was published in 2024 in the journal Cancer & Metabolism, titled Real-time assessment of relative mitochondrial ATP synthesis response against inhibiting and stimulating substrates (MitoRaise).

Research Process and Results

1. Development and Validation of MitoRaise Technology

The core of MitoRaise technology lies in assessing mitochondrial ATP synthesis rates by monitoring changes in ATP levels in real time. The research team designed an experimental workflow based on fluorescence signals, using a plasma membrane permeabilizer (PMP) to make cell membranes permeable, allowing substrates to enter cells and react with mitochondria. The specific steps are as follows:

• Cell Membrane Permeabilization: Cells were treated with 5-10 nM PMP to ensure membrane permeabilization while maintaining mitochondrial membrane integrity. Permeabilization was verified using trypan blue staining, ensuring that over 90% of cells were permeabilized.
• ATP Synthesis Rate Measurement: Permeabilized cells were seeded into a 96-well plate, and mitochondrial assay buffer (MAS buffer) containing adenylate kinase inhibitor (AP5A) was added, followed by ADP and a fluorescent substrate (ATPlite solution). Changes in fluorescence signals were used to monitor ATP levels in real time.
• Substrate Stimulation and Inhibition: After measuring basal ATP levels, stimulating substrates (e.g., glutamate and malate) and inhibiting substrates (e.g., rotenone and malonate) were sequentially added to measure the increase and decrease in ATP synthesis rates, respectively.

The research team validated the sensitivity and specificity of MitoRaise through a series of experiments. Results showed that MitoRaise could accurately measure changes in mitochondrial ATP synthesis rates under various conditions, including different quantities of mitochondria, varying cell numbers, mitochondria-damaged cells, and heterogeneous cell mixtures.

2. Analysis of Mitochondrial Function in Cell Lines

To further evaluate the potential applications of MitoRaise, the research team conducted a comprehensive analysis of mitochondrial function in multiple breast cancer cell lines and other cell lines. Experimental results revealed significant differences in total ATP levels, ATP synthesis rates (ASR), and mitochondrial DNA copy numbers (mtDNA-CN) among different cell lines. Notably, cell lines such as MCF7 and BT-474 exhibited higher mitochondrial respiratory activity, while MDA-MB-231 and BT-20 showed higher glycolytic activity.

Through correlation analysis, the team found that total ATP levels positively correlated with substrate-induced ATP synthesis rates in certain cell lines (e.g., MCF7 and BT-474), but not in others (e.g., MDA-MB-231 and BT-20). This suggests that MitoRaise can effectively distinguish between cells relying on oxidative phosphorylation and those relying on glycolysis.

3. Application in Clinical Samples

The research team also used MitoRaise to analyze peripheral blood mononuclear cells (PBMCs) from 19 breast cancer patients and 23 healthy women. Results showed that PBMCs from breast cancer patients exhibited lower basal ATP levels, rotenone response, malonate response, and mitochondrial DNA copy numbers, while glutamate-induced ATP synthesis rates were significantly higher compared to healthy controls. These findings indicate significant differences in mitochondrial metabolic status between breast cancer patients and healthy individuals.

Additionally, the team found that age negatively correlated with basal ATP levels, rotenone response, malonate response, and mitochondrial DNA copy numbers, suggesting a decline in mitochondrial function with aging.

Conclusions and Significance

MitoRaise technology provides a new method for assessing mitochondrial function by monitoring ATP synthesis rates in real time. This technology not only accurately measures dynamic changes in mitochondrial ATP synthesis but also distinguishes between cells with different metabolic types, demonstrating broad application potential. Particularly in research on mitochondrial metabolism in diseases such as cancer, aging, chronic fatigue syndrome, and type 2 diabetes, MitoRaise is expected to become an important tool.

Research Highlights

• Real-Time Monitoring: MitoRaise enables real-time measurement of mitochondrial ATP synthesis rates, overcoming the limitations of traditional methods.
• High Sensitivity and Specificity: Through a series of validation experiments, MitoRaise demonstrated high sensitivity and specificity under various conditions.
• Clinical Application Potential: Its application in PBMCs from breast cancer patients showed that MitoRaise can effectively distinguish mitochondrial metabolic differences in disease states.

Additional Valuable Information

The research team also noted that further optimization of MitoRaise requires combining oxygen consumption rate measurements to accurately calculate the phosphate-to-oxygen ratio (P/O ratio), thereby providing a more comprehensive assessment of mitochondrial function. Additionally, future studies will recruit participants from different age groups to further validate the relationship between mitochondrial metabolic status, aging, and disease.

MitoRaise technology offers a new tool for mitochondrial research and is expected to play a significant role in disease diagnosis and treatment.