Effects of tamoxifen inducible MerCreMer on gene expression in cardiac myocytes in mice

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 (Myh6-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.

Keywords

Transcriptome / gene expression / tamoxifen / Cre recombinase / MerCreMer / interferon / inflammation / TP53

References

[1] Fukushige S.Genomic targeting with a positive-selection lox integration vector allows highly reproducible gene expression in mammalian cells.Proc Natl Acad Sci U S A1992;89:7905-9 PMCID:PMC49823

[2] Agah R,French BA,Overbeek PA.Gene recombination in postmitotic cells. Targeted expression of Cre recombinase provokes cardiac-restricted, site-specific rearrangement in adult ventricular muscle in vivo.J Clin Invest1997;100:169-79 PMCID:PMC508177

[3] Pugach EK,Azofeifa JG,Leinwand LA.Prolonged Cre expression driven by the α-myosin heavy chain promoter can be cardiotoxic.J Mol Cell Cardiol2015;86:54-61 PMCID:PMC4558343

[4] Metzger D,Chiba H.Conditional site-specific recombination in mammalian cells using a ligand-dependent chimeric Cre recombinase.Proc Natl Acad Sci U S A1995;92:6991-5 PMCID:PMC41457

[5] Feil R,Mascrez B,Metzger D.Ligand-activated site-specific recombination in mice.Proc Natl Acad Sci U S A1996;93:10887-90 PMCID:PMC38252

[6] Sohal DS,Crackower MA.Temporally regulated and tissue-specific gene manipulations in the adult and embryonic heart using a tamoxifen-inducible Cre protein.Circ Res2001;89:20-5

[7] Koitabashi N,Zaiman AL.Avoidance of transient cardiomyopathy in cardiomyocyte-targeted tamoxifen-induced MerCreMer gene deletion models.Circ Res2009;105:12-5 PMCID:PMC2747596

[8] Hall ME,Hall JE.Systolic dysfunction in cardiac-specific ligand-inducible MerCreMer transgenic mice.Am J Physiol Heart Circ Physiol2011;301:H253-60 PMCID:PMC3129917

[9] Heinen A,Flögel U.4-hydroxytamoxifen does not deteriorate cardiac function in cardiomyocyte-specific MerCreMer transgenic mice.Basic Res Cardiol2021;116:8 PMCID:PMC7864833

[10] Soonpaa MH.Survey of studies examining mammalian cardiomyocyte DNA synthesis.Circ Res1998;83:15-26

[11] Cheedipudi SM,Fan S.Exercise restores dysregulated gene expression in a mouse model of arrhythmogenic cardiomyopathy.Cardiovasc Res2020;116:1199-213 PMCID:PMC7177479

[12] Auguste G,Lombardi R,Willerson JT.Suppression of activated FOXO transcription factors in the heart prolongs survival in a mouse model of laminopathies.Circ Res2018;122:678-92 PMCID:PMC5834384

[13] Chen SN,Karmouch J.DNA damage response/TP53 pathway is activated and contributes to the pathogenesis of dilated cardiomyopathy associated with LMNA (Lamin A/C) mutations.Circ Res2019;124:856-73 PMCID:PMC6460911

[14] Wingett SW.FastQ Screen: a tool for multi-genome mapping and quality control.F1000Res2018;7:1338 PMCID:PMC6124377

[15] Dobin A,Schlesinger F.STAR: ultrafast universal RNA-seq aligner.Bioinformatics2013;29:15-21 PMCID:PMC3530905

[16] Risso D,Speed TP.Normalization of RNA-seq data using factor analysis of control genes or samples.Nat Biotechnol2014;32:896-902 PMCID:PMC4404308

[17] Love MI,Anders S.Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.Genome Biol2014;15:550 PMCID:PMC4302049

[18] Auguste G,Matkovich SJ.BET bromodomain inhibition attenuates cardiac phenotype in myocyte-specific lamin A/C-deficient mice.J Clin Invest2020;130:4740-58 PMCID:PMC7456228

[19] Rouhi L,Chen SN.Haploinsufficiency of Tmem43 in cardiac myocytes activates the DNA damage response pathway leading to a late-onset senescence-associated pro-fibrotic cardiomyopathy.Cardiovasc Res2021;117:2377-94

[20] Pépin G,Höning K.Cre-dependent DNA recombination activates a STING-dependent innate immune response.Nucleic Acids Res2016;44:5356-64 PMCID:PMC4914124

[21] Thyagarajan B,Groth A.Mammalian genomes contain active recombinase recognition sites.Gene2000;244:47-54

[22] Loonstra A,Beverloo HB.Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells.Proc Natl Acad Sci U S A2001;98:9209-14 PMCID:PMC55399