Abstract Body: Background:
Heart disease and cancer represent leading causes of death in the United States, sharing significant overlap in risk factors. A major clinical challenge is that off-target cardiotoxicity is associated with various chemotherapeutics, including doxorubicin (DOX), an indispensable first-line anti-cancer agent for many cancers. Previous efforts have established the mechanism by which DOX causes tumor cytotoxicity, mainly via inhibition of DNA replication in cancer cells. However, how DOX causes cytotoxicity in non-replicative cardiomyocytes remains elusive. One potential explanation centers on rapid uptake of DOX within mitochondria of cardiomyocytes, causing aberrant mitochondrial function and cardiomyocyte cell death.

Hypothesis:
To this end, we hypothesize that mitochondrial accumulation of DOX triggers the mitochondrial unfolded protein response (UPRmt), which plays a critical role in mediating DOX-induced cardiotoxicity within cardiomyocytes. We also posit that genetic manipulation of this pathway may serve as a potential therapeutic means by which to ameliorate DOX cardiotoxicity.

Approach:
To test this hypothesis, we employed human cardiac (AC-16) cells subjected to increasing doses of DOX for 12hrs (0mM, 0.5mM, and 1mM), with subsequent isolation of RNA and protein to assess UPRmt gene activation using RT-qPCR and western blot analysis (n=3), respectively. In parallel, C57BL/6 mice (n=7 per group) were dosed with either saline or DOX at 10mg/kg, i.p., with whole hearts collected to investigate the activation of the UPRmt at the transcript and protein levels. Likewise, using AC-16 cells with reduced eIF2a levels, an upstream regulator of the UPRmt, we repeated DOX treatment (n=3) to assess for increased cytotoxicity using LIVE/DEAD staining on flow cytometry.

Results/Conclusions:
Our results showed a significant induction of UPRmt genes at both the transcript (~2-fold increase in ATF5, CLPP, CLPX, LONP1 in cells and mice; p≤0.05) and protein levels (~2-fold increase in CLPP, CLPX and 1.5-fold increase in HSPA9 in mice; p≤0.05) using both in-vitro and in-vivo models of DOX cardiotoxicity. Importantly, we found that shRNA knock-down of eIF2a blunted the UPRmt response, and exacerbated DOX-induced cytotoxicity (p≤0.05) compared to DOX treated scramble control cells. These findings establish the UPRmt as a bonafide mediator of DOX cardiotoxicity, supporting the rationale for therapeutically targeting this pathway to mitigate DOX cardiotoxicity.