Abstract Body: Autophagy, the degradation and recycling of intracellular materials, plays an important role in cardiomyopathy and heart failure. In the heart, either hyperactivation or inhibition of autophagy can lead to cell death and organ dysfunction. We previously developed dual-fluorescent Autophagy Detecting Nanoparticle (ADN), a molecular imaging probe designed to track autophagosome fate, monitoring autophagy levels in vitro and in whole-body imaging studies in vivo. We hypothesize that a deeper real-time analysis of cardiomyocyte autophagy is warranted.
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) expressing autophagy protein Light Chain 3 (LC3)-eGFP were generated using a sequential GSK3/Wnt signaling inhibitors protocol. ADN was synthesized and characterized in-house per our previous publications. Live cell and immunofluorescence were imaged by confocal microscopy. Sarcomeres stained with alpha-actinin antibody were quantified using ImageJ. Autophagy and troponin proteins were measured by Western blots.
The hiPSC-CMs spontaneously contract in culture, with organized sarcomeres (~1.8 µm in lengths), and form significantly increased eGFP-LC3 puncta upon autophagy activation by starvation or 0.5 µM rapamycin after 24 hours. Throughout the two-week differentiation process, we observe increased expression of autophagy markers LC3, Beclin1, and p62 in the first two days relative to the contracting hiPSC-CMs. Troponin expression begins on day 6 and increases until day 14. ADN uptake and activation was significantly increased in hiPSC-CMs with autophagy activated, colocalizing with eGFP-LC3 puncta and with >80% precision-recall. Conversely, autophagy inhibition by bafilomycin or chloroquine significantly reduced ADN activation. Furthermore, cardiotoxic doxorubicin (2 µM, 24 hours) impaired ADN signal in hiPSC-CMs but had no effect on sarcomere integrity.
To the best of our knowledge, this is the first report studying the role of autophagy and autophagy modulation in hiPSC-CMs. We further validated the real-time detection of hiPSC-CMs autophagy by ADN. This imaging platform opens novel portals into preclinical drug discovery, allowing beneficial autophagy modulation to be defined, enhancing CM survival.