Mitochondrial Targeting Application of Genetically Encoded Voltage Indicators (GEVIs)

Background and Research Motivation

Mitochondria, as the energy factories of eukaryotic cells, play crucial roles in various cellular processes, including bioenergetic conversion, metabolite synthesis, cell survival, calcium storage, and heat production. In organs requiring high aerobic metabolism, such as the brain and heart, normal mitochondrial function is particularly critical. Maintaining the resting membrane potential of neurons and cardiomyocytes consumes a large amount of energy, mainly achieved through the sodium-potassium pump (Na+/K+ ATPase). The inner mitochondrial membrane contains carriers, ion channels, and ion pumps responsible for transporting various substances, thereby generating and affecting the mitochondrial membrane potential (MMP, ψm). ψm is related to various cellular physiologies, including energy production, reactive oxygen species (ROS) formation, and promoting transmembrane transport of metabolic substrates and ions. Additionally, ψm influences mitochondrial morphology and participates in processes such as mitophagy and apoptosis.

Current methods for measuring mitochondrial membrane potential (Δψm) rely on the distribution of lipophilic cation dyes. However, there are no genetically encoded fluorescent indicators (GEVIs) specifically for MMP. This study attempts to address this gap by screening and developing GEVIs that can target mitochondria and monitor dynamic changes in their membrane potential.

Research Source and Author Information

This article was written by Run-Zhou Yang, Dian-Dian Wang, Sen-Miao Li, Pei-Pei Liu, and Jian-Sheng Kang, from institutions including the Laboratory of Clinical Systems Biology at the First Affiliated Hospital of Zhengzhou University, the Department of Neurology at the First Affiliated Hospital of Zhengzhou University, and the School of Medicine at Zhengzhou University. The research was published in Neurosci. Bull. in 2024.

Research Process and Methods

Research Subjects and Experimental Design

The study first selected four GEVIs, including Arclight and ASAP1 derived from GFP, and Somoarchon and PROPS derived from rhodopsin. These GEVIs were targeted to mitochondria by fusing a mitochondrial targeting signal sequence (mt, 4cox8) to their N-terminus. The study evaluated the mitochondrial targeting efficiency of these GEVIs and verified their voltage sensitivity through cellular experiments.

Cell Culture and Transfection

The experiments used HEK293T, COS-7, and HeLa cells, which were cultured in Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum and grown at 37°C in a 5% CO2/95% air environment. For short-term expression, cells were transfected using the calcium phosphate precipitation method.

Cell Imaging and Colocalization Analysis

Imaging was performed using a laser scanning confocal microscope, and colocalization analysis was conducted using ImageJ software, quantifying Pearson’s correlation coefficient to evaluate mitochondrial targeting efficiency.

Isolation and Culture of Primary Cardiomyocytes and Neurons

Heart and hippocampal tissues from 0-day-old C57BL mice were dissected, digested with trypsin and collagenase, and further dissociated using polished pipettes. Cells were then plated on cover slips coated with Matrigel. Cardiomyocytes began spontaneous beating after 24 hours, while hippocampal cells were further dissociated and centrifuged to obtain single cells before plating and culturing.

Virus Preparation and Transduction

Adeno-associated virus (AAV) and lentivirus were used to transduce cardiomyocytes and neurons. Viruses were prepared by transfecting HEK293T cells using the calcium phosphate precipitation method, and extracted using freeze-thaw cycles.

Construction of Stable Transfected Cell Lines

Stable transfected cell lines were constructed through lentiviral transduction and subsequent screening. Cells were grown in media containing thiostrepton, and the presence of plasmid sequences was verified by PCR and sequencing.

Electrophysiology and Voltage Imaging

Whole-cell patch-clamp recordings were performed at room temperature using a custom optical system with a data acquisition rate of 10 kHz. Cell activity was recorded in voltage-clamp mode, and the camera was set to trigger mode to capture voltage pulses.

Live Cell Imaging

Cells were imaged under a fluorescence microscope in Tyrode’s buffer, with images acquired in streaming mode. To capture spontaneous voltage fluctuations, images were acquired in streaming mode.

In Vitro Screening System

A T5 promoter and Shine-Dalgarno sequence were inserted into the pcDNA3.1(-) vector to enable expression in both prokaryotic and mammalian cells. Bacterial clones with high fluorescence were screened by PCR and sequence analysis for further analysis.

Experimental Results

Screening and Verification

In HeLa or COS-7 cells, the study found that among the GEVIs, mt-ASAP1 showed the highest mitochondrial targeting efficiency and demonstrated voltage sensitivity. Additionally, mt-ASAP1 was found to be sensitive to ROS in cardiomyocytes. Through various mutations, the study developed the ASAP3-st mutant with high voltage sensitivity and insensitivity to ROS.

Reduction of Mitochondrial Membrane Potential

During anesthesia, fiber photometry experiments revealed that anesthesia caused mitochondrial depolarization, which mt-ASAP3-st could effectively monitor.

Conclusion

The study successfully developed four GEVIs that can target mitochondria and verified their voltage sensitivity and ROS sensitivity in various cell types. The results contribute to further understanding the role of mitochondria in different physiological and pathological processes and provide a high-throughput screening tool for developing new drugs to treat mitochondrial dysfunction.

Research Highlights

1. Developed four mitochondria-targeted genetically encoded voltage indicators (MPI-1 to MPI-4) and verified their application in various cell types.
2. Discovered and addressed the ROS sensitivity issue of mt-ASAP1 in cardiomyocytes by developing the ROS-insensitive mt-ASAP3-st through mutations.
3. For the first time, monitored anesthesia-induced mitochondrial membrane potential depolarization in vivo using fiber photometry, providing new insights into the effects of anesthetic drugs on mitochondrial function.

Research Significance

This study not only enriches the tools for monitoring mitochondrial membrane potential but also provides new means for in-depth research on physiological and pathological processes related to energy metabolism. These GEVIs have broad potential applications in drug screening, disease model research, and basic biological studies.