Abstract
Doxorubicin is a potent and widely used antineoplastic agent. However, almost 30% of patients treated with doxorubicin develop heart failure. The mechanism behind its cardiotoxic effect is elusive. Recent evidence suggests that doxorubicin accumulates in cardiac mitochondria causing both mutations and reduction in number of mitochondrial DNA copies. This scenario reduces the expression of mitochondrial proteins encoded by mitochondria DNA, but not the expression of nuclear-encoded mitochondrial proteins, which leads to mitonuclear protein imbalance and mitochondrial dysfunction. It has been demonstrated that changes in mitochondrial proteostasis trigger the mitochondrial retrograde signaling, a communication pathway between mitochondria and nucleus that maintains mitochondrial quality upon stress conditions. In the present study, we aim to evaluate the impact of doxorubicin on both cardiac mitonuclear protein imbalance and activation of mitochondrial retrograde signaling. We expect that acute doxorubicin treatment will activate the mitochondrial retrograde signaling in attempt to reestablish mitochondrial proteostasis. However, chronic doxorubicin treatment (similar to the clinical practice) will disrupt this mitochondrial quality control mechanism and results in heart failure. To test this hypothesis, c57/bl6 mice will be treated with doxorubicin at 10 mg/kg. The acute effect of doxorubicin will be measured at 2, 12, 24 and 48 hours after doxorubicin injection. To determine the chronic effect of doxorubicin, mice will receive 3 doses (10 mg/kg) of doxorubicin every 48 hours. Measurements will be performed 11 days after the first doxorubicin injection. We will analyze life span, cardiac function, mitonuclear protein imbalance, accumulation of mitochondrial misfolded proteins, mitochondrial bioenergetics, mitochondrial H2O2 release and mitochondrial retrograde signaling activation (gene expression of hspa9, hsp-60 and clpp). The hormetic effect of doxorubicin in activating mitochondrial retrograde signaling will be validated in isolated adult cardiomyocytes after individual knocking-down of genes involved in the mitochondrial retrograde signaling (hspa9, hsp-60 and clpp). We will analyze cell shortening, Ca+2 transient and viability. Finally, we will use a SJ4100 C. elegans strain expressing a GFP-Hsp-6 construct to track in real time the impact of doxorubicin on mitochondrial retrograde signaling. GFP fluorescence will be measured to determine the mitochondrial retrograde signaling activation. In addition, a cause-effect relationship between doxorubicin-induced toxicity and mitochondrial retrograde signaling will be studied in C. elegans with loss of function for proteins involved in mitochondrial retrograde signaling. We will measure mitochondrial respiration, reactive oxygen species production, mitonuclear protein imbalance, misfolded protein accumulation, pharyngeal pump and life span. Our preliminary findings show that acute doxorubicin treatment causes a transient cardiac dysfunction in rodents, which is associated with impaired mitochondrial metabolism. Additionally, doxorubicin treatment activates mitochondrial retrograde signaling in C. elegans. These results support our initial hypothesis. Relevance: Understanding the role of mitochondrial retrograde signaling may help in developing better strategies to treat cancer. (AU)
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