Effects of Tamoxifen Inducible MerCreMer on Gene Expression in Cardiac Myocytes in Mice

Overview

Journal J Cardiovasc Aging

Date 2022 Jan 26

PMID 35079750

Authors

Leila Rouhi

Siyang Fan

Sirisha M Cheedipudi

Melis Olcum

Hyun-Hwan Jeong

Zhongming Zhao

Priyatansh Gurha

Ali J Marian

Affiliations

Soon will be listed here.

Abstract

The Cre-LoxP technology, including the tamoxifen (TAM) inducible MerCreMer (MCM), is increasingly used to delineate gene function, understand the disease mechanisms, and test therapeutic interventions. We set to determine the effects of TAM-MCM on cardiac myocyte transcriptome. Expression of the MCM was induced specifically in cardiac myocytes upon injection of TAM to myosin heavy chain 6-MCM () mice for 5 consecutive days. Cardiac function, myocardial histology, and gene expression (RNA-sequencing) were analyzed 2 weeks after TAM injection. A total of 346 protein coding genes (168 up- and 178 down-regulated) were differentially expressed. Transcript levels of 85 genes, analyzed by a reverse transcription-polymerase chain reaction in independent samples, correlated with changes in the RNA-sequencing data. The differentially expressed genes were modestly enriched for genes involved in the interferon response and the tumor protein 53 (TP53) pathways. The changes in gene expression were relatively small and mostly transient and had no discernible effects on cardiac function, myocardial fibrosis, and apoptosis or induction of double-stranded DNA breaks. Thus, TAM-inducible activation of MCM alters cardiac myocytes gene expression, provoking modest and transient interferon and DNA damage responses without exerting other discernible phenotypic effects. Thus, the effects of TAM-MCM on gene expression should be considered in discerning the bona fide changes that result from the targeting of the gene of interest.

Citing Articles

Causes and consequences of DNA double-stranded breaks in cardiovascular disease.

Marian A Mol Cell Biochem. 2024; .

PMID: 39404936 DOI: 10.1007/s11010-024-05131-9.

Prolonged tamoxifen-enriched diet is associated with cardiomyopathy and nutritional frailty in mice.

Halpage J, DaSilva Pantoja P, Mancarella S Exp Physiol. 2024; 109(4):513-523.

PMID: 38291801 PMC: 10984784. DOI: 10.1113/EP091668.

Genetic inactivation of β-catenin is salubrious, whereas its activation is deleterious in desmoplakin cardiomyopathy.

Olcum M, Fan S, Rouhi L, Cheedipudi S, Cathcart B, Jeong H Cardiovasc Res. 2023; 119(17):2712-2728.

PMID: 37625794 PMC: 11032201. DOI: 10.1093/cvr/cvad137.

Metformin protects against pulmonary hypertension-induced right ventricular dysfunction in an age- and sex-specific manner independent of cardiac AMPK.

McNair B, Polson S, Shorthill S, Yusifov A, Walker L, Weiser-Evans M Am J Physiol Heart Circ Physiol. 2023; 325(2):H278-H292.

PMID: 37389952 PMC: 10393374. DOI: 10.1152/ajpheart.00124.2023.

Cardiomyocyte-Specific Wt1 Is Involved in Cardiac Metabolism and Response to Damage.

Diaz Del Moral S, Benaouicha M, Villa Del Campo C, Torres M, Wagner N, Wagner K J Cardiovasc Dev Dis. 2023; 10(5).

PMID: 37233178 PMC: 10219250. DOI: 10.3390/jcdd10050211.

References

Loonstra A, Vooijs M, Beverloo H, Allak B, van Drunen E, Kanaar R . Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells. Proc Natl Acad Sci U S A. 2001; 98(16):9209-14. PMC: 55399. DOI: 10.1073/pnas.161269798. View

Agah R, Frenkel P, French B, Michael L, Overbeek P, Schneider M . Gene recombination in postmitotic cells. Targeted expression of Cre recombinase provokes cardiac-restricted, site-specific rearrangement in adult ventricular muscle in vivo. J Clin Invest. 1997; 100(1):169-79. PMC: 508177. DOI: 10.1172/JCI119509. View

Rouhi L, Cheedipudi S, Chen S, Fan S, Lombardi R, Chen X . Haploinsufficiency of Tmem43 in cardiac myocytes activates the DNA damage response pathway leading to a late-onset senescence-associated pro-fibrotic cardiomyopathy. Cardiovasc Res. 2020; 117(11):2377-2394. PMC: 8861264. DOI: 10.1093/cvr/cvaa300. View

Sohal D, Nghiem M, Crackower M, Witt S, Kimball T, Tymitz K . Temporally regulated and tissue-specific gene manipulations in the adult and embryonic heart using a tamoxifen-inducible Cre protein. Circ Res. 2001; 89(1):20-5. DOI: 10.1161/hh1301.092687. View

Thyagarajan B, Guimaraes M, Groth A, Calos M . Mammalian genomes contain active recombinase recognition sites. Gene. 2000; 244(1-2):47-54. DOI: 10.1016/s0378-1119(00)00008-1. View

Auguste G, Gurha P, Lombardi R, Coarfa C, Willerson J, Marian A . Suppression of Activated FOXO Transcription Factors in the Heart Prolongs Survival in a Mouse Model of Laminopathies. Circ Res. 2018; 122(5):678-692. PMC: 5834384. DOI: 10.1161/CIRCRESAHA.117.312052. View

Pepin G, Ferrand J, Honing K, Jayasekara W, Cain J, Behlke M . Cre-dependent DNA recombination activates a STING-dependent innate immune response. Nucleic Acids Res. 2016; 44(11):5356-64. PMC: 4914124. DOI: 10.1093/nar/gkw405. View

Heinen A, Godecke S, Flogel U, Miklos D, Bottermann K, Spychala A . 4-hydroxytamoxifen does not deteriorate cardiac function in cardiomyocyte-specific MerCreMer transgenic mice. Basic Res Cardiol. 2021; 116(1):8. PMC: 7864833. DOI: 10.1007/s00395-020-00841-9. View

Feil R, Brocard J, Mascrez B, Lemeur M, Metzger D, Chambon P . Ligand-activated site-specific recombination in mice. Proc Natl Acad Sci U S A. 1996; 93(20):10887-90. PMC: 38252. DOI: 10.1073/pnas.93.20.10887. View

10.

Risso D, Ngai J, Speed T, Dudoit S . Normalization of RNA-seq data using factor analysis of control genes or samples. Nat Biotechnol. 2014; 32(9):896-902. PMC: 4404308. DOI: 10.1038/nbt.2931. View

11.

Pugach E, Richmond P, Azofeifa J, Dowell R, Leinwand L . Prolonged Cre expression driven by the α-myosin heavy chain promoter can be cardiotoxic. J Mol Cell Cardiol. 2015; 86:54-61. PMC: 4558343. DOI: 10.1016/j.yjmcc.2015.06.019. View

12.

Koitabashi N, Bedja D, Zaiman A, Pinto Y, Zhang M, Gabrielson K . Avoidance of transient cardiomyopathy in cardiomyocyte-targeted tamoxifen-induced MerCreMer gene deletion models. Circ Res. 2009; 105(1):12-5. PMC: 2747596. DOI: 10.1161/CIRCRESAHA.109.198416. View

13.

Cheedipudi S, Hu J, Fan S, Yuan P, Karmouch J, Czernuszewicz G . Exercise restores dysregulated gene expression in a mouse model of arrhythmogenic cardiomyopathy. Cardiovasc Res. 2019; 116(6):1199-1213. PMC: 7177479. DOI: 10.1093/cvr/cvz199. View

14.

Hall M, Smith G, Hall J, Stec D . Systolic dysfunction in cardiac-specific ligand-inducible MerCreMer transgenic mice. Am J Physiol Heart Circ Physiol. 2011; 301(1):H253-60. PMC: 3129917. DOI: 10.1152/ajpheart.00786.2010. View

15.

Love M, Huber W, Anders S . Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014; 15(12):550. PMC: 4302049. DOI: 10.1186/s13059-014-0550-8. View

16.

Chen S, Lombardi R, Karmouch J, Tsai J, Czernuszewicz G, Taylor M . DNA Damage Response/TP53 Pathway Is Activated and Contributes to the Pathogenesis of Dilated Cardiomyopathy Associated With LMNA (Lamin A/C) Mutations. Circ Res. 2019; 124(6):856-873. PMC: 6460911. DOI: 10.1161/CIRCRESAHA.118.314238. View

17.

Fukushige S, Sauer B . Genomic targeting with a positive-selection lox integration vector allows highly reproducible gene expression in mammalian cells. Proc Natl Acad Sci U S A. 1992; 89(17):7905-9. PMC: 49823. DOI: 10.1073/pnas.89.17.7905. View

18.

Metzger D, Clifford J, Chiba H, Chambon P . Conditional site-specific recombination in mammalian cells using a ligand-dependent chimeric Cre recombinase. Proc Natl Acad Sci U S A. 1995; 92(15):6991-5. PMC: 41457. DOI: 10.1073/pnas.92.15.6991. View

19.

Dobin A, Davis C, Schlesinger F, Drenkow J, Zaleski C, Jha S . STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2012; 29(1):15-21. PMC: 3530905. DOI: 10.1093/bioinformatics/bts635. View

20.

Auguste G, Rouhi L, Matkovich S, Coarfa C, Robertson M, Czernuszewicz G . BET bromodomain inhibition attenuates cardiac phenotype in myocyte-specific lamin A/C-deficient mice. J Clin Invest. 2020; 130(9):4740-4758. PMC: 7456228. DOI: 10.1172/JCI135922. View