4-hydroxytamoxifen does not deteriorate cardiac function in cardiomyocyte-specific MerCreMer transgenic mice

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.

Keywords: 4-hydroxytamoxifen; Cardiac energetics; Cardiac function; Cardiomyopathy; aMHC-MerCreMer/loxP system.

Fig. 1

OH-Txf causes efficient gene deletion in floxed αMHC-MerCreMer mice. Representative western blots and summarized data of a Akt1 and Akt2, b GSK-3β, and c p38 of hearts from iCM-Akt1/2KO, iCM-GSK3βKO, and iCM-p38KO mice, respectively, are shown. Cardiomyocyte restricted gene deletion was initiated by intraperitoneal application of 20 mg/kg OH-Txf on 5, 7 or 10 consecutive days as indicated. d Representative immunofluorescence stainings of cross sectioned (upper panel) and longitudinal slices (lower panel) of hearts from iCM-Akt1/2 knock out mice (KO) or WT mice are shown. Administration of 20 mg/kg OH-Txf for five consecutive days resulted in loss of both Akt1 (left panel) and Akt2 (right panel) in cardiomyocytes. Data are normalized to WT values and presented as mean ± SD. *p < 0.05 vs WT (unpaired, two-tailed student’s T-test)

Fig. 2

OH-Txf treatment does disturb neither cardiac function nor cardiac energetic status in αMHC-MerCreMer mice. a Experimental protocol; αMHC-MerCreMer mice received 20 mg/kg 4-hydroxytamoxifen (OH-Txf) intraperitoneally for 5 consecutive days. Data were obtained before (d0) and 5, 10, 15, and 19 days after the first OH-Txf injection, respectively. b Cardiac function was determined by MR imaging. An example image (left), and summarized data of cardiac output (CO), ejection fraction (EF), and end diastolic volume (EDV) are shown. c Cardiac energy status was determined using ³¹P MR spectroscopy. MR spectra were obtained from a voxel of the left ventricular free wall (yellow square in the example image; left). Example spectra from d0, d5, and d19 (middle), and summarized data of PCr/ATP ratios (right) are shown. d Creatine formation was assessed by CrCEST; example images from d0, d5, and d19 (left), and summarized data (right) are shown. Data are presented as mean ± SD. n = 4; *p < 0.05 vs d0 (one-way repeated-measure ANOVA, followed by Tukey’s post hoc test)

Fig. 3

Tissue characterization of OH-Txf treatment does not induce tissue fibrosis or cause lipid accumulation and/or tissue oedema a Example T1 maps of hearts from OH-Txf treated αMHC-MerCreMer mice at d0, d5, and d19 (left), and summarized data of mean T1 relaxation time (right) are shown. b Cardiac fibrosis was assessed by Masson trichrome staining (left) or immunohistological collagen III staining (right) at d19 of untreated (-OH-Tamoxifen) or OH-Txf treated (+ OH-Tamoxifen) hearts of αMHC-MerCreMer mice. Example images for Masson trichrome staining and collagen III staining as well as summarized data of collagen III content (right) are shown. Scale bare represents 50 µm (Masson trichrome) or 200 µm (collagen III). c Example T2 maps of hearts from OH-Txf treated αMHC-MerCreMer mice at d0, d5, and d19 (left), and summarized data of mean T2 relaxation time (right) are shown (n = 4). d Relative mRNA expression of Nppa, Nppb and Atp2a2 were determined as marker genes for cardiomyopathy by qPCR in hearts of αMHC-MerCreMer mice at d19 after OH-Txf treatment for 5 days (Cre + OH-Txf + ; n = 5), and Cre-negative mice that did not receive OH-Txf (Cre- OH-Txf-; n = 6). The results were normalized to Actb mRNA expression, and x-fold induction was calculated relative to the expression in the Cre- OH-Txf- group. Data are presented as mean ± SD. *p < 0.05 vs d0 (one-way repeated-measure ANOVA, followed by Tukey’s post hoc test) or unpaired, two-tailed student’s T-test

Fig. 4

Long-term effects of OH-Txf treatment on cardiac function in αMHC-MerCreMer mice. a Experimental protocol; αMHC-MerCreMer mice received 20 mg/kg 4-hydroxytamoxifen (OH-Txf) intraperitoneally for 5 consecutive days. Cardiac function was analysed by echocardiography before (d0) and 21 and 42 days after the first OH-Txf injection, respectively. b Representative parasternal short-axis M-mode views of d0 (left), d21 (mid), and d42 (right) recordings. Scaling of the x-axis in seconds, scaling of the y-axis: mm. c Summarized data for cardiac output (CO), ejection fraction (EF), end diastolic volume (EDV), and stroke volume (SV). Data are presented as mean ± SD. n = 5; *p < 0.05 vs d0 (one-way repeated-measure ANOVA, followed by Tukey’s post hoc test)

Fig. 5

4-Hydroxytamoxifen treatment for 10 days does not induce cardiomyopathy. a Experimental protocol; αMHC-MerCreMer mice received 20 mg/kg 4-hydroxytamoxifen (OH-Txf) intraperitoneally for 10 consecutive days. Cardiac function was analysed by echocardiography before (d0) and 7, 14, 21 and 42 days after the first OH-Txf injection, respectively. b Representative parasternal short-axis M-mode views of d0 (left), d7 (mid), and d42 (right) recordings. Scaling of the x-axis in seconds, scaling of the y-axis: mm. c Summarized data for cardiac output (CO), ejection fraction (EF), end diastolic volume (EDV), and stroke volume (SV). Data are presented as mean ± SD. n = 4; *p < 0.05 vs d0 (one-way repeated-measure ANOVA, followed by Tukey’s post hoc test)