Quantitative approach to assess function and apoptosis changes in the H9C2 cardiomyocyte cell line following differentiation and Doxorubicin exposure

Introduction: For some decades, the anthracycline doxorubicin (DOX) has been used in the treatment of solid tumors and leukemia. It is one of the most widely and successfully applied chemotherapeutic drugs and over the last 20 years has increased the 5-year survival rates for pediatric cancer patients from 58% to 80%. However, the clinical utility and dosage regimens of DOX are often limited by severe side effects.The most severe side effect of DOX is chronic dilated cardiomyopathy (DCM). Chronic DCM leads to cardiac dysfunction and eventually congestive heart failure that without heart transplant may result in death. Chronic DCM occurs in about 10-20% of cancer survivors and is often manifest 10-15 years following treatment. The molecular mechanisms of disease progression after this long latency remains to be determined however DOX associated reactive oxygen species (ROS) generation has been considered to play a major role in this late toxicity. While not ruling out a contribution for ROS, antioxidant therapy has not however been successful in reducing the incidence of cardiotoxicity in patients treated with DOX. Some studies have suggested that apoptosis is involved in doxorubicin-induced cardiotoxicity although little evidence of substantial apoptosis was reported in heart biopsies of patients with DCM who had previously been treated with DOX. Given that cardiac toxicity arises only years after treatment, a low level of apoptosis may however be sufficient for the DCM phenotype observed. With this in mind, we investigated the mechanisms of toxicity of DOX and whether its toxic effect involved apoptosis.Hypothesis and aims: The aim of this study was to understand the mechanisms underlying DOX induced late cardiotoxicity. We hypothesized that, DOX sensitization of cardiomyocytes during chemotherapy leads to their lower resistance to a subsequent apoptotic stimulus. We postulated that this increased sensitivity was due to a DOX-induced dedifferentiation of cardiomyocytes, which are relatively resistant to apoptosis, into a cardiomyoblast phenotype that is more sensitive to death-inducing stimulus. To validate our hypothesis, we first compared the sensitivity to apoptosis of H9C2 cardiomyocytes (H9C2 CM) with that of H9C2 cardiomyoblasts. Secondly, to gain insight into the delayed action of DOX in vivo, we tested the effect of preconditioning cells to DOX upon their sensitivity to subsequent DOX treatment. The degree of differentiation and the mechanisms underlying apoptosis were probed in the contexts of these aims.Findings: H9C2 CM show greater resistance to apoptosis than H9C2, and using a non-biased proteomics approach, RT-qPCR as well as an immunoblotting assay, proteins of the apoptotic machinery that might underlie this resistance were identified. In particular, we found that anti-apoptotic proteins XIAP was increased upon differentiation. However, we surprisingly found that pro-apoptotic proteins caspase-8, caspase-3 and FAS were increased upon differentiation. H9C2 CM preconditioned to DOX exhibit signs of partial dedifferentiation. However, preconditioned H9C2 CM exposed to an apoptotic stimulus exhibited increased resistance to apoptosis. Together, these findings shed new light on the cell biology of DOX-induced toxicity and is consistent with the hypothesis that cardiomyocyte dedifferentiation initially protects against cell death.

Jaar van publicatie:2019