Abstract 3385: Cardiac Stem Cell Survival During DNA Damage Is Regulated By Modulation Of Histone Acetyl Transferase p300
Background: Many sources of oxidative stress, including ischemia/reperfusion and chemotherapy, can induce DNA damage in the heart, leading to loss of cardiomyocytes and development of heart failure. Cardiac stem cells (CSCs) may serve to regenerate injured myocardium. However, ischemic injury has been associated with senescence and apoptosis in CSCs, potentially limiting their contribution to tissue repair. The histone acetyltransferase p300 has been shown to be an important regulator of genome stability and is involved in cell survival in response to DNA damage. We hypothesized that p300 could play a role in CSC response to oxidant stress.
Methods: c-kit+, sca-1+ CSCs were isolated and cloned by serial dilution from adult mouse hearts. CSC clones were stably transfected with lentivirus vectors carrying shRNAmir against p300 or a non-silencing RNA (NS) and maintained under puromycin selection prior to treatment. CSCs were exposed to doxorubicin (DOX), a DNA-damaging agent, for defined intervals. Levels of p300 were monitored by Western analysis and expression of DNA damage-regulated genes was assessed using multiplex realtime QPCR. CSCs were treated with 1 uM DOX and survival was assessed over a period of 72h.
Results: Levels of p300 increased rapidly in CSCs within 4h after exposure to doxorubicin and were sustained for at least 24h. CSCs expressing p300 shRNAmir (p300 KD) contained ~20% of the amount of p300 in CSCs transfected with the NS sequence (NS CSCs). QPCR analysis of DNA repair and cell cycle control genes indicated that 44 were significantly expressed in CSCs; 13 of these were up-regulated by p300 loss, and 5 were down-regulated, indicative of a DNA damage response to p300 loss.
Both cell proliferation rates and progression through the cell cycle were slower in p300 KD vs. NS CSCs. Surprisingly, following 48h treatment with DOX, p300 KD CSCs had 32% improved survival vs. NS CSCs.
Conclusions: Under acute genotoxic stress, p300 accumulates in CSCs. Reducing p300 levels induces the expression of genes involved in the DNA damage response, probably as a compensatory response, and slows CSC proliferation. Loss or inhibition of p300 is likely to cause multifactorial growth arrest in CSCs and thereby reduce their vulnerability to genotoxic agents.
This research has received full or partial funding support from the American Heart Association, AHA National Center.