1Division of Oncology, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA.
2Heart, Lung and Vascular Institute, Division of Cardiovascular Health and Disease, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA.
Correspondence to: Dr. Abitha Sukumaran, Division of Oncology, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, 3333 Burnet Avenue, Cincinnati, OH 45229, USA. E-mail: email@example.com ;
© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, sharing, adaptation, distribution and reproduction in any medium or format, for any purpose, even commercially, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
Cardiomyopathy, heart failure, gene regulation
In this issue of The Journal of Cardiovascular Aging, Rouhi et al. have determined the effects of tamoxifen (TAM) and MerCreMer on cardiomyocyte transcriptome, cardiac function, and histopathology at an early developmental stage in mice.
The Cre-loxP system is a powerful and versatile tool to control site-specific recombination of mammalian genomic DNA. Site-specific Cre recombinase-mediated DNA recombination allows for the conditional control of gene expression within transgenic animals in a tissue-specific manner by employing a promoter known to be expressed specifically in such tissue of interest. More specifically, the gene of interest is flanked (floxed) by two loxP (locus of x-over, P1) sites in the presence of Cre recombinase, which then catalyzes the site-specific recombination of DNA between those loxP sites, leading to tissue-specific gene editing. However, the approach is flawed by the lack of control over the timing of Cre recombinase expression which often parallels the expression of the chosen promoter. Consequently, Cre-mediated, tissue-specific gene ablation could lead to embryonic, fetal, or neonatal lethality. Since the heart is the first organ to develop and start functioning during embryogenesis, the noted risks rule out examining gene function at later developmental stages. To circumvent the risks, conditional gene manipulation developed in animal models allows for temporal control over the expression of a transgene. Using the MerCreMer system, in particular, Cre recombinase is fused to mutant estrogen-receptor ligand-binding domains on either side (MerCreMer). This mutant estrogen receptor is insensitive to estrogen, but sensitive to TAM, an estrogen antagonist used to control the expression of MerCreMer.
Typically, cardiac-specific genetic disruption is achieved through the α-myosin heavy chain (α-MHC) promoter-driven MerCreMer expression (Mhy6-MerCreMer). This mouse model was first developed by Sohal et al.. Although this model has been widely used to temporally control gene expression at the tissue-specific level, the possible side effects of TAM and/or MerCreMer remain controversial, thus potentially limiting the application of this approach. Here, Rouhi et al. show the effects of TAM and MerCreMer on cardiac structure and function in mice at four weeks of age, as assessed by echocardiography. Phenotypic changes, such as myocardial fibrosis, apoptosis, and DNA damage, were determined by histological examination of the myocardium and Western blotting. They found only small and transient changes in gene expression in response to TAM and MerCreMer, but no remarkable changes in cardiac structure or function.
The original work by Sohal et al. on TAM inducible Mhy6-MerCreMer mice reported no adverse effects on cardiac function, but some other studies did report transient cardiac dysfunction, dysregulated energy metabolism and cardiomyopathy[1,3-11], mainly attributable to MerCreMer expression and TAM dosage. A study by Koitabashi et al. showed that TAM-induced nuclear translocation of MerCreMer resulted in transient, but severe cardiomyopathy, independent of the presence of loxP gene, thus directly pointing to MerCreMer and TAM. They associated this outcome with a significant decrease in some proteins involved in cardiac bioenergetics machinery and calcium handling, but these effects were transient and found to normalize fully upon recovery. Finally, they showed that TAM may play a role in the cardiac dysfunction seen in these mice. Other groups have corroborated these findings[4,6]. Studies have also shown that moderate to high doses of TAM in Mhy6-MerCreMer mice induced DNA damage, leading to heart failure and death. Therefore, the authors concluded that 30 μg/g body weight of TAM resulted in maximum recombination and minimum cardiac toxicity. Another study reported the presence of focal fibrosis and depressed left-ventricular function in TAM-inducible MHC-MerCreMer mice. This condition was found to be present regardless of the transgene. Furthermore, TAM injection induced proinflammatory cytokines, such as IL-6, TNF-α, IL-1β, IFNγ and CCL2. Markers of cardiac hypertrophy, such as ANF, BNP and COL3A1, were also found to be increased in these mice. These results prompted the authors to highlight the need to include age-matched TAM-injected MerCreMer mice as controls. In contrast, a recent work by Heinen et al. showed that using 4-hydroxyTAM (OH-Tam), a metabolite of TAM, did not induce adverse cardiotoxicity in Mhy6-MerCreMer mice.
In this study, Rouhi et al. studied changes in the transcriptome profile of cardiac myocytes in response to TAM and MerCreMer. TAM (30 mg/kg/day) was injected subcutaneously for five consecutive days from day 14 after birth. This time point was chosen as cardiomyocytes completely stop proliferating by this age. The dose was chosen for its reported minimal cardiac toxicity[6,7]. The authors performed cardiac function, myocardium histology, and mRNA expression (RNA-seq) studies 14 days after the last TAM injection. No discernible changes were noted in either myocardial histology or cardiac function, only small changes in mRNA expression in response to interferon and tumor protein 53 (TP53) pathways [Figure 1]. No significant changes were reported in the mRNA expression of cardiomyocyte transcriptome in response to TAM and MerCreMer at six months of age, suggesting that the observed mild changes in mRNA expression were transient.
Figure 1. Effects of tamoxifen and MerCreMer on cardiac function, phenotype and transcriptome. Tamoxifen inducible MerCreMer expression did not cause changes in cardiac function and histology in mice at four weeks of age. Nonetheless, small, but transient changes in cardiac transcriptome in response to tamoxifen and MerCreMer expression were observed. (A) Tamoxifen was injected at 14 days to Myh6-MerCreMer mice. Wild-type mice lacking Myh6-MerCreMer were used as controls. (B) Cardiac function was assessed by echocardiography at four weeks of age prior to the sacrifice of mice. Hearts were harvested to determine cardiac fibrosis, apoptosis and DNA damage. Cardiac transcriptome profile was also assessed in the hearts of these mice. (C) Modest, but transient increases in interferon response and TP53 pathway were observed.
From the above discussion, it will be recalled that the use of MerCreMer mice, temporally controlled by TAM, was developed to circumvent the risks associated with conventional CreLoxP gene disruption. At the same time, however, the possible side effects of TAM and/or MerCreMer remain controversial with conflicting studies. The authors emphasized the use of Mhy6-MerCreMer mice treated with TAM as a control, comparing to studying the gene of interest. However, the effects of TAM injection and activation of MerCreMer on cardiac function and myocardial histology at later stages, i.e., after four weeks (period chosen in this study), were not addressed. Therefore, in view of cardiac pathology, it will be necessary to conduct a time-course study of changes in the cardiac transcriptome between four weeks up to six months of age to determine the duration of changes in mRNA expression. We expect that the authors will address these limitations in future studies.
Conceived and drafted the manuscript: Sukumaran A, Sadayappan SAvailability of data and materials
Not applicable.Financial support and sponsorship
Dr. Sadayappan has received support from National Institutes of Health grants (R01 HL130356, R01 HL105826, R01 AR078001, and R01 HL143490), American Heart Association, Institutional Undergraduate Student (19UFEL34380251), Transformation (19TPA34830084) awards, the PLN Foundation (PLN crazy idea) awards, as well as Novo Nordisk, AstraZeneca, MyoKardia, Merck and Amgen.Conflicts of interest
Dr. Sadayappan provided consulting and collaborative research studies to the Leducq Foundation (CURE-PLAN), Red Saree Inc., Greater Cincinnati Tamil Sangam, AavantiBio, Pfizer, Novo Nordisk, AstraZeneca, MyoKardia, Merck and Amgen. Dr. Sukumaran declared that there are no conflicts of interest.Ethical approval and consent to participate
Not applicable.Consent for publication
© The Author(s) 2022.
1. Rouhi L, Fan S, Cheedipudi SM, et al. Effects of tamoxifen inducible MerCreMer on gene expression in cardiac myocytes in mice. J Cardiovasc Aging 2022;2:8.DOIPubMed PMC
2. Sohal DS, Nghiem M, Crackower MA, et al. Temporally regulated and tissue-specific gene manipulations in the adult and embryonic heart using a tamoxifen-inducible Cre protein. Circ Res 2001;89:20-5.DOIPubMed
3. Heinen A, Gödecke S, Flögel U, et al. 4-hydroxytamoxifen does not deteriorate cardiac function in cardiomyocyte-specific MerCreMer transgenic mice. Basic Res Cardiol 2021;116:8.DOIPubMed PMC
4. Asp ML, Martindale JJ, Metzger JM. Direct, differential effects of tamoxifen, 4-hydroxytamoxifen, and raloxifene on cardiac myocyte contractility and calcium handling. PLoS One 2013;8:e78768.DOIPubMed PMC
5. Bersell K, Choudhury S, Mollova M, et al. Moderate and high amounts of tamoxifen in αMHC-MerCreMer mice induce a DNA damage response, leading to heart failure and death. Dis Model Mech 2013;6:1459-69.DOIPubMed PMC
6. Hall ME, Smith G, Hall JE, Stec DE. Systolic dysfunction in cardiac-specific ligand-inducible MerCreMer transgenic mice. Am J Physiol Heart Circ Physiol 2011;301:H253-60.DOIPubMed PMC
7. Koitabashi N, Bedja D, Zaiman AL, et al. Avoidance of transient cardiomyopathy in cardiomyocyte-targeted tamoxifen-induced MerCreMer gene deletion models. Circ Res 2009;105:12-5.DOIPubMed PMC
8. Lexow J, Poggioli T, Sarathchandra P, Santini MP, Rosenthal N. Cardiac fibrosis in mice expressing an inducible myocardial-specific Cre driver. Dis Model Mech 2013;6:1470-6.DOIPubMed PMC
9. Pugach EK, Richmond PA, Azofeifa JG, Dowell RD, Leinwand LA. Prolonged Cre expression driven by the α-myosin heavy chain promoter can be cardiotoxic. J Mol Cell Cardiol 2015;86:54-61.DOIPubMed PMC
10. Yan J, Sultana N, Zhang L, et al. Generation of a tamoxifen inducible Tnnt2MerCreMer knock-in mouse model for cardiac studies. Genesis 2015;53:377-86.DOIPubMed PMC
11. Yan J, Zhang L, Sultana N, et al. A murine myh6mercremer knock-in allele specifically mediates temporal genetic deletion in cardiomyocytes after tamoxifen induction. PLoS One 2015;10:e0133472.DOIPubMed PMC
Sukumaran A, Sadayappan S. Optimization of tamoxifen-induced gene regulation in cardiovascular research. J Cardiovasc Aging 2022;2:21. http://dx.doi.org/10.20517/jca.2022.12