Doxorubicin causes depletion of cardiac primitive cell pool that may add to the mechanisms of doxorubicin-mediated delayed cardiotoxicity.

Doxorubicin is one of the most active anti-cancer drugs, widely used for the treatment of several malignancies, however its clinical implementation is limited by high incidence of cardiovascular events, most serious of them being the cardiomyopathy followed by congestive heart failure. Known studies of the mechanisms of doxorubicin toxicity focused on cardiomyocyte damage. But then cardiac primitive cells are considered to provide the adult heart with a substantial growth reserve determining the function of the heart throughout life. The role of this cell population in the myocardial response to different pathologic stimuli has been documented in animals and humans, suggesting cardiac primitive cells as possible pathophysiologic target in cardiac diseases. We advance the hypothesis that cardiotoxicity of doxorubicin may be due to its effects on cardiac primitive cells. Hence, we examined doxorubicin toxicity on this cell population. CD117-positive cells isolated from adult human normal atria were incubated with increasing concentrations of doxorubicin hydrochloride (0.1, 0.5 and 1μM), followed by the evaluation of proliferation and apoptosis after 12, 24, 48 and 60 hours. Proliferation was evaluated by BrdU incorporation and its rate dropped from 12.84±1.83% (control, n=4) to 2.41±0.27% (24 hours, 1μM, n=4, p<0,05). Immunoblotting of proteins from cardiac primitive cells lysate indicated the upregulation of cyclin B1 paired with reduced expression of phospho-cdk1 and increased expression of p27, corresponding to the presence of cell cycle block at G2/M transition. The expression of phospho-p53 (Ser15) the ratio phosphop53/total p53 increased 2.1-fold and 1.2-fold (p<0,05), respectively. Apoptosis was evidentiated by the detection of early mitochondrial potential disruption, followed by 2,4-fold increase in caspase-3 activity (4.8x10e-3 μmol pnitroaniline/min/ml in control vs 11.5x10e-3 at 48 hours). With respect to control, apoptotic cells were 6.2-fold and 10.4-fold (p<0,05) more numerous after 12 hours and 48 hours of incubation with 1μM of doxorubicin, respectively. Taking into consideration the known mechanism of doxorubicin cardiotoxicity, namely oxidative stress, we observed reduced expression of catalase and manganese superoxide dismutase in cardiac primitive cells after 36 hours of incubation with 1μM doxorubicin. In conclusion, doxorubicin has profound effects on cardiac primitive cell proliferation and survival. The depletion of CD117-positive cardiac primitive cell pool may add to the mechanisms of doxorubicin-mediated delayed cardiotoxicity.