Abstract
Conditional, cell-type-specific transgenic mouse lines are of high value in cardiovascular research. A standard tool for cardiomyocyte-restricted DNA editing is the αMHC-MerCreMer/loxP system. However, there is an ongoing debate on the occurrence of cardiac side effects caused by unspecific Cre activity or related to tamoxifen/oil overload. Here, we investigated potential adverse effects of DNA editing by the αMHC-MerCreMer/loxP system in combination with a low-dose treatment protocol with the tamoxifen metabolite 4-hydroxytamoxifen (OH-Txf). αMHC-MerCreMer mice received intraperitoneally OH-Txf (20 mg/kg) for 5 or 10 days. These treatment protocols were highly efficient to induce DNA editing in adult mouse hearts. Multi-parametric magnetic resonance imaging revealed neither transient nor permanent effects on cardiac function during or up to 19 days after 5 day OH-Txf treatment. Furthermore, OH-Txf did not affect cardiac phosphocreatine/ATP ratios assessed by in vivo 31P MR spectroscopy, indicating no Cre-mediated side effects on cardiac energy status. No MRI-based indication for the development of cardiac fibrosis was found as mean T1 relaxation time was unchanged. Histological analysis of myocardial collagen III content after OH-Txf confirmed this result. Last, mean T2 relaxation time was not altered after Txf treatment suggesting no pronounced cardiac lipid accumulation or tissue oedema. In additional experiments, cardiac function was assessed for up to 42 days to investigate potential delayed side effects of OH-Txf treatment. Neither 5- nor 10-day treatment resulted in a depression of cardiac function. Efficient cardiomyocyte-restricted DNA editing that is free of unwanted side effects on cardiac function, energetics or fibrosis can be achieved in adult mice when the αMHC-MerCreMer/loxP system is activated by the tamoxifen metabolite OH-Txf.
Highlights
Conditional gene modification technologies are widely used in the field of cardiovascular research to study physiological and pathophysiological gene functions in mouse models
Whereas the original work from the Molkentin group investigating the αMHCMerCreMer/loxP system does not show detrimental effects on cardiac function [31], other studies report on the occurrence of systolic dysfunction, disturbed cardiac energetics, and cardiomyopathy, which were dependent on Cre-activation [12, 20]
OH-Txf in combination with the αMHC-MerCreMer/loxP system is an effective method for inducible, cardiomyocyte-restricted DNA editing in adult mice
Summary
Conditional gene modification technologies are widely used in the field of cardiovascular research to study physiological and pathophysiological gene functions in mouse models. The point mutations alter the ligand-binding properties in that the mutated sites have a higher affinity to synthetic ligands, such as tamoxifen, and a reduced affinity for the natural ligand oestrogen. In addition to this temporal control, the use of tissue-specific promotors to drive inducible Cre expression in mice with loxP-containing genes allows spatial control of mutagenesis. The α-myosin heavy chain promotor (αMHCMerCreMer) allows cardiomyocyte-restricted DNA editing [31] This αMHC-MerCreMer/loxP system is used in many research laboratories, there is an ongoing debate on potential cardiac site effects that would limit the applicability of this gene-editing strategy. Because OH-Txf is the active metabolite of tamoxifen which has a much higher affinity to the oestrogen receptor compared to tamoxifen, the question raises whether cardiac side effects can be reduced or avoided using lower doses of OH-Txf compared to tamoxifen
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