(28f) Optogenetic-Mediated Preconditioning As a Novel Approach to Protect Cells from Stress-Induced Injury

Stem cell-based cardiac repair faces a significant challenge of low viability and retention of transplanted cells in the stressful recipient heart environment. Recent evidence indicates that mitochondrial membrane potential (MMP) plays a crucial role in preconditioning, where low levels of stress can reduce subsequent damage. However, current methods for modulating MMP are inadequate and limit the ability to track its mechanism in cytoprotection.

To overcome these limitations, our lab developed a next-generation mitochondrial-targeted optogenetic technology (mOpto) by expressing light-gated channelrhodopsin proteins specifically in the inner membrane of mitochondria. We hypothesized that mOpto-mediated mild and transient MMP depolarization could protect cells against stress-induced injury. To test this hypothesis, AC16 cells expressing the mOpto gene were illuminated with short-term, low-intensity (3 hours, 0.2 mW/mm²) blue light (470nm) to induce mild transient MMP depolarization, followed by exposing to various stressors such as FCCP (20 μM, 24 hours), H₂O₂ (200 μM, 24 hours), or ischemia-reperfusion (I2/R4: glucose-free medium and 0% O₂ for 2 hours, and complete culture medium and oxygenated for 4 hours). At the end of the experiment, cell viability, ROS (reactive oxygen species) levels, and membrane potential were measured to determine the preconditioning-mediated cytoprotective effect. Additionally, we added mitoQ, a mitochondrial-specific antioxidant, to the preconditioning cells to examine the involvement of ROS in mOpto-mediated preconditioning.

Our results showed that MMP recovered completely within the light intensity range used for preconditioning, indicating its reversibility. We also observed significantly higher cell viability in the preconditioning group compared to the control groups in I2/R4, along with reduced ROS levels and partially recovered MMP. Similarly, preconditioning improved cell injury and mitochondrial dysfunction in FCCP and H₂O₂ treated cells. Finally, mitoQ had negligible effects on mOpto-mediated cytoprotective effects.

In summary, our study revealed that mOpto-mediated mild and transient mitochondrial depolarization effectively protects cells from various types of stress. Furthermore, our results suggest that the cytoprotective pathway engaged in this process is not dependent on ROS signaling. Although future mechanistic studies are warranted, our findings highlight the potential of mOpto-mediated preconditioning as a promising strategy to enhance the therapeutic value of engineered cells and deepen our understanding of cellular stress responses.