Dr Chen presented his findings on the role of autophagy in myocardial ischaemia–reperfusion (IR) injury based on a novel molecular imaging technique in a pre-clinical pig model. He began by highlighting that cardiomyocyte death is a hallmark of acute MI (AMI). He provided background by describing the three types of cellular death pathways (apoptosis, necrosis and autophagy), and the complex cross-talk between these cell death mechanisms.1 Apoptosis is programmed, while necrosis is an irreversible cell death. Autophagy is an evolutionarily conserved catabolic cellular process, during which cells digest organelles in their cytoplasm and recycle the constituents. Autophagy is thought to play an important role in many cardiovascular diseases. The pathophysiological impact of autophagy has been shown to promote cardiomyocyte death or recovery. A major barrier in the study of autophagy is the inability to accurately detect and quantify autophagosomes within the area at risk during AMI.
Dr Chen proposed that in vivo imaging may aid in quantification and provide mechanistic insights of these different cell death pathways during AMI. He noted that the annexin V-magnetic nanoparticle AnxCLIO-Cy5.5 was previously demonstrated to detect apoptosis in vivo by MRI with good spatial resolution in a mouse heart following AMI.2–4 More recently, a gadolinium–thiazole-based nanoparticle, which binds to the exposed DNA of ruptured cardiomyocytes, was demonstrated to detect necrosis in vivo by MRI in mouse heart following AMI.5–7
He stated that fluorescence imaging allows multiplexing, and thus plays an important part in understanding the fate of individual cells through the cell death pathways. Likewise, multiplexed imaging using nanoparticle probes is feasible and reveals additional insights, suggesting that early cardiomyocyte apoptosis may be reversible.
Dr Chen described in detail the molecular events during autophagy and emphasised that an expanded lysosomal compartment upon formation of the autolysosome is a hallmark of autophagy.8 He described two autophagy imaging constructs that reflect lysosomal enzyme (cathepsin) activity: cathepsin-activatable fluorochrome (CAF) and autophagy-detecting nanoparticle (ADN).9,10 ADN was rationally designed for enhanced sensitivity of autophagy detection and is based on a Food and Drug Administration (FDA)-approved drug, Feraheme (ferumoxytol), which is surface decorated with CAF peptides. The advantages of the ADN probe include dual near-infrared fluorescent and MRI readouts.
Dr Chen presented the results of ADN imaging of cardiomyocyte autophagy in a mouse model involving 24-hour starvation. Compared to the fed control mice, autophagy was induced in the starved mice and was detected by increased uptake of the ADN probe, both by MRI and fluorescent imaging. Systemic profiling of the ADN fluorescence showed increased autophagy (ADN levels) in the heart, spleen and small intestine compared to other organs, such as the lung, kidney or liver.
Next, Dr Chen presented his current hypothesis that the ADN, based on ferumoxytol, used to target lysosomal compartments during autophagy in vivo, could provide a robust fluorescent readout of autophagy levels in animal models of AMI. This hypothesis was tested using both mouse and pig models of IR injury.
Mice were subjected to 35 minutes of ischaemia by ligation of the left coronary artery, followed by 4 hours of reperfusion. At the onset of reperfusion, both ADN and CAF (internal control) probes were co-injected intravenously via the tail vein for ex vivo imaging. The results showed that ADN detects autophagy in IR mice (n=5) and is far more sensitive than CAF (n=5). Importantly, ADN activation was specific and localised in the ischaemic myocardium.
Treatment with rapamycin, a robust activator of autophagy through inhibition of the protein kinase mammalian target of rapamycin, led to a reduction in apoptosis by 23% and infarct size by 45%, thus providing molecular insights on the role of autophagy.10
In the pig model of AMI, adult swine were subjected to 2 hours of ischaemia by left anterior descending artery occlusion, followed by 30 minutes of mechanical support with Impella or extracorporeal membrane oxygenation (ECMO) and finally 2 hours of reperfusion. The ADN probe was injected intracoronary during reperfusion for ex vivo imaging. As expected from the results of the mouse model, a significant increase in ADN signal was seen in the area at risk compared to the uninjured (septal) myocardium. Of note, reperfusion significantly increased autophagosome formation (indicated by light chain 3B-II levels), which was attenuated by Impella (n=4), but not by ECMO (n=4).
In conclusion, this study demonstrates the feasibility of quantitative autophagy imaging in the heart following AMI and shows that unloading with Impella and ECMO differentially impacts autophagy. Future studies will focus on characterising additional molecular readouts of autophagy in unloaded swine hearts after AMI to further advance the translation of the novel autophagy imaging technology.