Monitoring Autophagy in the Heart and in Tumors Treated with Potentially Cardiotoxic Chemotherapy

The treatment of cancer has been revolutionized by the development of targeted and immune-based therapies.
However, many of these new therapies, as well as established ones such as doxorubicin, can damage the heart
and cause severe heart failure. A growing body of evidence suggests that perturbations in cardiomyocyte
autophagy may play a central role in chemotherapy-induced heart failure, and the ability to image this process
in vivo could provide important insights. We have recently developed an autophagy-detecting nanoparticle (ADN)
and have used this agent to image autophagy in the heart during chemotherapy with doxorubicin and dasatanib.
The core of the agent consists of ferumoxytol, an MRI-detectable nanoparticle, to which several polyarginine-
Cy5.5 moieties are attached. The polyarginine peptides facilitate the translocation of the agent into the cell,
where it is taken up by autophagosomes and trafficked to the lysosomes. Folding of the polyarginine peptides
also results in stacking and quenching of the Cy5.5 fluorochromes until the peptides are cleaved by cathepsins
in the autophagolysosomes. We have shown using chemical inhibitors/stimulators of autophagy, and cells with
genetic deletion of the key autophagy proteins ATG5 and ATG7, that the activation of ADN is specific for
autophagy. Likewise, using a transgenic mouse with overexpression of the DDiT4L transcription factor in the
heart and the canonical autophagy model of intermittent fasting, we have shown that ADN can detect autophagy
in vivo with a high degree of sensitivity and specificity. In this proposal we will use ADN to interrogate changes
in autophagy in both the heart and cancer. While the upregulation of autophagy in the heart is protective, it has
the potential to either protect or harm tumors undergoing chemotherapy. We hypothesize that the imaging of
autophagy with ADN will allow conditions and strategies to be identified where the upregulation of autophagy is
cardioprotective but does not attenuate the effect of the chemotherapy on the tumor. In aim 1 of the proposal,
we will make a small chemical modification to the probe, enabling it to be detected before and after its activation,
and use confocal/super-resolution microscopy of live cells to develop a detailed kinetic model of autophagy.
IPSC-derived cardiomyocytes and relevant tumor cell lines will be studied. In aim 2 we will assess the impact of
autophagy upregulation on a library of tumor cell lines exposed to a large panel of chemotherapies, many known
to cause heart failure. In aim 3 we will use a multispectral fluorescence approach to image autophagy, apoptosis
and inflammation in the heart and in tumors in mice in vivo. The impact of autophagy upregulation will be
assessed and a detailed kinetic model of autophagy in the heart and in tumors will be derived. Execution of the
proposed aims will provide important insights into the role of autophagy in the heart and cancer, and provide a
platform to identify safe and effective autophagy regulating strategies to protect the heart during chemotherapy.

Grant Number: 5R01HL166810-03
NIH Institute/Center: NIH
Principal Investigator: Howard Chen