Hello, my name is Isabella Church and I am a second year osteopathic medical student at New York Institute of Technology, College of Osteopathic Medicine, located in Old Westbury on Long Island, New York. I'm excited to be here presenting my poster titled AMPK Beta 1 Knockdown Increases Autophagy Flux in Doxorubicin Induced H9C2 Cardiomyocyte Death. Doxorubicin, or DOX, is a widely used chemotherapeutic for broad-spectrum treatment of solid tumors and leukemias. However, DOX is associated with severe dose-dependent cardiotoxic effects that culminate in irreversible congestive heart failure, and this significantly limits its use. Although the exact mechanism for this is unknown, AMP-activated protein kinase, or AMPK, has been implicated in the process. AMPK is a heterotrimeric protein kinase composed of a catalytic alpha subunit and two regulatory subunits, a beta and a gamma, each with multiple isoforms. Previously, it was suggested that AMPK reduced DOX toxicity. However, data from our lab showed marked reduction in DOX-induced cardiac injury in mice with AMPK-alpha-2 knockout and exacerbation of toxicity with MK-8722, which is a pan-AMPK activator. This led us to hypothesize that the AMPK subunits have differential roles in regulating DOX cardiotoxicity. In this study, we focused primarily on AMPK beta-1 and beta-2 isoforms and defining their functional roles in regulating autophagy and cardiomyocyte death. So moving on to my methods. We did this by siRNA knockdown of the individual subunits and assessing their impact on cell death and autophagy flux. So in designing our study, there were three main goals we were trying to achieve. First is effective and specific knockdown of the individual subunits. Second is evaluating how these subunits are regulating autophagy. This is done by measuring LC3B levels, which is an autophagosome marker. But because the autophagosome itself gets degraded, we need to compare uninhibited to inhibited levels. So we do this by treating cells with protease inhibitors, pepsatin A and E64D. Our third goal is to evaluate how autophagy is regulating cell death in doxorubicin treatment. So we did this by treating cells with one micromolar of doxorubicin for 24 hours and measuring global cell death via propidium iodide staining and apoptotic cell death via the marker cleave CasPase 3. Moving on to my results. So going back to our first goal, we want to evaluate effective and specific knockdown of the AMPK beta 1 and beta 2 subunits. So starting on the left here in this western blot, in the center panel, showing levels of AMPK beta 1 and beta 2. And as you can see here, both are present in our control samples. And we achieved effective knockdown of beta 1 and beta 2 individually with relatively minimal knockdown of their opposite. So this indicated that our methods are both effective and specific. Next we evaluated autophagy flux by treating cells with protease inhibitors and comparing uninhibited to inhibited samples. So down here in this bottom panel, LC3-1 and LC3-2. LC3-1 is found in the cytosol, whereas LC3-2 is found on the autophagosome and is more indicative of autophagy flux. In the samples with AMPK beta 1 down regulation, you can see that the levels of LC3-2 significantly increased in the presence of inhibitors, suggesting enhanced autophagy flux in the absence of AMPK beta 1. Moving over to the right here, I'm showing cell death by measuring cleave caspase 3 and PI staining. And so in the western blot, we can see here that there is minimal cell death with knockdown of the subunits alone, which is supported by the PI data. In our dox treated samples, you can see increased cell death and beta 1 and beta 2 knockdown compared to control. And this is supported by the PI data for beta 1 knockdown. However, the PI data for beta 2 knockdown showed no significant difference from control. So this is something we're going to have to evaluate further. In addition, beta 2 did not exhibit any dramatic change in autophagy flux compared to control. So once again, further evaluation of beta 2 is needed. And that brings me to my conclusions and future directions. So as I just discussed, we can see that AMPK beta 1 knockdown increases autophagy flux in H9C2 cardiomyocytes, and this is associated with increased dox cardiotoxicity. Second, these results contrast the prevailing view that AMPK promotes autophagy, and this is cardioprotective against the toxic effects of dr. Rubicin. We saw here that the removal of AMPK beta 1 is what promotes autophagy and increases cell death. So we can conclude that AMPK beta 1 decreases autophagy and is cardioprotective. In this sense, our results support our hypothesis that each of the specific AMPK subunit isoforms have a unique and distinctive role in regulating autophagy and cell death. In terms of our future work, a lot of our current experiments are aimed at replicating and validating these results. While I've repeated some of these studies and gotten similar results, we are further increasing our sample size to answer our questions more definitively. We are also using other markers of cell death and damage to further evaluate the impact of these subunits on cardiotoxicity. In addition, we are currently exploring the role of the beta subunit on regulating mitophagy, which is autophagy within the mitochondria specifically, as well as the effect of dox treatment on both autophagy and mitophagy. All these studies are being replicated in human cardiomyocytes, specifically the AC16 cell line, to confirm our findings and make them more relevant for clinical application. Lastly, this work is part of a much larger project aimed at evaluating the other AMPK subunits, so alpha and gamma, to evaluate their role in mediating cardiotoxicity and autophagy and mitophagy. To conclude, I just want to touch on the potential impact of this work and its relevance to clinical medicine. As I mentioned before, doxorubicin is a widely used chemotherapeutic, but its efficacy in treating cancer has been reduced due to its irreversible cardiotoxic effects. And so it is our hope that these findings and our future studies could be used to inform protective strategies against doxorubicin-induced cardiotoxicity in cancer patients, while preserving the drug's antitumor efficacy. So this concludes my poster presentation. Thank you very much for your time and attention.

AMPK subunits

doxorubicin cardiotoxicity

autophagy

cardiomyocyte death

cancer treatment strategies