Numerous mouse models have utilized Cre/loxP technology to modify gene expression. Adverse effects of Cre recombinase activity have been reported, including in the heart. However, the mechanisms associated with cardiac Cre toxicity are largely unknown. Here, we show that expression of Cre in cardiomyocytes induces a DNA damage response, resulting in cardiomyocyte apoptosis and cardiac fibrosis and dysfunction. In an effort to increase the recombination efficiency of a widely used tamoxifen-sensitive Cre transgene under control of the α-myosin heavy chain promoter (α-MHC-MerCreMer), we observed myocardial dysfunction and decreased survival, which were dependent on the dose of tamoxifen injected. After excluding a Cre-independent contribution by tamoxifen, we found that Cre induced myocardial fibrosis, activation of pro-fibrotic genes, and cardiomyocyte apoptosis. Examination of the molecular mechanisms showed activation of DNA damage response signalling and p53 stabilization in the absence of loxP sites, suggesting that Cre induced illegitimate DNA breaks. Cardiomyocyte apoptosis was also induced by expressing Cre using adenoviral tranduction, indicating that the effect was not dependent on genomic integration of the transgene. Cre-mediated homologous recombination at loxP sites was dose-dependent and had a ceiling effect at approximately 80% of cardiomyocytes showing recombination. By titrating the amount of tamoxifen to maximize recombination while minimizing animal lethality, we determined that 30 μg/gm/day of tamoxifen for three consecutive days is the optimal condition for the α-MHC-MerCreMer system to induce recombination in the Rosa26-lacZ strain. Our results further highlight the importance of experimental design and of including appropriate genetic controls for Cre expression.
- Received June 22, 2012.
- Accepted July 29, 2013.
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