Persistent effects of doxorubicin on cardiac gene expression

Abstract

During administration of the anthracycline antitumour agents, their cardiotoxicity can progress from cardiac dysfunction to heart failure. Cardiomyopathy can also develop years after receiving anthracyclines. To determine if persistent and/or progressive anthracycline effect(s) are referable to anthracycline effects on cardiac gene expression, steady-state mRNA levels were determined 4 days (n=8), 4 weeks (n=7) and 10 weeks (n=7) after doxorubicin (DOX; 2 mg/kg IV) in a well-characterized rabbit model. Levels of mRNA for alpha -actin, beta -myosin heavy chain and the calcium pump of the sarcoplasmic reticulum (SERCA2a) in the left ventricle (LV) were determined by Northern blot hybridization and expressed relative to an 18S constitutive marker. The mRNA levels for the high molecular weight subunit (cardiac isoform) of the ryanodine receptor (RyR2), sarcolemmal calcium channel (dihydropyridine receptor; DHPR), angiotensin-converting enzyme (ACE), angiotensin II receptor (ATR) and atrial naturetic peptide prohormone (ANP) were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analysis, and expressed relative to GAPDH, a constitutive marker. Histopathologic evidence for anthracycline-induced myocardial cell injury was absent (score <1) in all hearts examined except one (score=1.1; 4 weeks post-DOX), which was considered separately. Relative mRNA levels for beta -myosin heavy chain 4 days after DOX increased 1.9-fold compared to the vehicle-treated group, but by 4 weeks levels had returned to baseline. Relative mRNA levels for DHPR were increased 1.2-fold 4 days after DOX and were persistently increased 1.9- and 2.2-fold 4 and 10 weeks after DOX, respectively. The mRNA levels for ANP were first decreased (4.5-fold) 4 days after DOX. Four weeks after DOX, ANP message levels approached Control in seven out of eight rabbits. The one rabbit with early LV histopathology 4 weeks post-DOX had increased mRNA for DHPR (2.7-fold) and ANP (80-fold). Between 4 and 10 weeks after DOX, mRNA levels for ANP increased C 16-fold: evidence for late progression. In situ hybridization with specific riboprobes localized the persistent increase in DHPR and the progressive increase in ANP to myocytes. Thus, DOX alters steady-state mRNA levels in LV that are referable to both persistent and progressive anthracycline effects on myocellular gene expression.