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. 2018 Sep 21;19(1):175.
doi: 10.1186/s12881-018-0670-1.

Muscle-specific regulation of right ventricular transcriptional responses to chronic hypoxia-induced hypertrophy by the muscle ring finger-1 (MuRF1) ubiquitin ligase in mice

Robert H Oakley  1 , Matthew J Campen  2 , Michael L Paffett  2 , Xin Chen  3 , Zhongjing Wang  4 , Traci L Parry  3 , Carolyn Hillhouse  3 , John A Cidlowski  1 , Monte S Willis  5   6   7
Affiliations

Muscle-specific regulation of right ventricular transcriptional responses to chronic hypoxia-induced hypertrophy by the muscle ring finger-1 (MuRF1) ubiquitin ligase in mice

Robert H Oakley et al. BMC Med Genet. .

Abstract

Background: We recently identified a role for the muscle-specific ubiquitin ligase MuRF1 in right-sided heart failure secondary to pulmonary hypertension induced by chronic hypoxia (CH). MuRF1-/- mice exposed to CH are resistant to right ventricular (RV) dysfunction whereas MuRF1 Tg + mice exhibit impaired function indicative of heart failure. The present study was undertaken to understand the underlying transcriptional alterations in the RV of MuRF1-/- and MuRF1 Tg + mice.

Methods: Microarray analysis was performed on RNA isolated from the RV of MuRF1-/-, MuRF1 Tg+, and wild-type control mice exposed to CH.

Results: MuRF1-/- RV differentially expressed 590 genes in response to CH. Analysis of the top 66 genes (> 2-fold or < - 2-fold) revealed significant associations with oxidoreductase, transcription regulation, and transmembrane component annotations. The significant genes had promoters enriched for HOXD12, HOXC13, and RREB-1 protein transcription factor binding sites. MuRF1 Tg + RV differentially expressed 150 genes in response to CH. Analysis of the top 45 genes (> 3-fold or < - 3-fold) revealed significant associations with oxidoreductase-metabolic, glycoprotein-transmembrane-integral proteins, and alternative splicing/splice variant annotations. The significant genes were enriched for promoters with ZIC1 protein transcription factor binding sites.

Conclusions: The differentially expressed genes in MuRF1-/- and MuRF1 Tg + RV after CH have common functional annotations related to oxidoreductase (including antioxidant) and transmembrane component functions. Moreover, the functionally-enhanced MuRF1-/- hearts regulate genes related to transcription, homeobox proteins, and kinases/phosphorylation. These studies also reveal potential indirect effects of MuRF1 through regulating Rreb-1, and they reveal mechanisms by which MuRF1 may transcriptionally regulate anti-oxidant systems in the face of right heart failure.

Keywords: Gene expression; Hypoxia; Microarray; MuRF1; Right heart failure.

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Conflict of interest statement

Ethics approval

Mice were bred at the University of North Carolina at Chapel Hill and all the hypoxia procedures were conducted at the University of New Mexico with full approval of the UNC-Chapel Hill and University of New Mexico Institutional Animal Care and Use Committees and were carried out in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Differential gene expression of MuRF1−/− right ventricle after three weeks CH exposure. a Summary of how differentially expressed genes in hypoxia-challenged MuRF1−/− RV tissue compared to MuRF1+/+ were identified. b Top 35 increased unique genes (> 2-fold) and top 31 decreased unique genes (<− 2-fold) compared to MuRF1+/+ hearts. Asterisk (*) designates same/related sequence for duplicate gene name. c Identification of transcription factors with binding sites of top 35 increased and top 31 decreased genes using TRANSFAC® FMatch. The number of genes in dataset and enrichment (sites/sequence ratio) for each transcription factor is calculated to the right. d STRING analysis of differentially expressed genes for known interactions and e STRING analysis pathway ID statistics. MuRF1−/−: N = 5 biological replicates; MuRF1+/+: N = 6 biological replicates
Fig. 2
Fig. 2
Functional annotation of differentially expressed genes in MuRF1−/− right ventricle after three weeks CH exposure. a. Hypercluster analysis of differentially expressed MuRF1−/− genes in the right ventricle after hypoxia challenge using the DAVID v6.8 Functional Annotation Bioinformatics database. Differentially expressed gene symbols are on the left, functional categories along the top (database source in parentheses). The cluster fold enrichment is color-coded (key in lower right). b. Reverse transcriptase quantitative PCR analysis of MuRF1−/− hearts of differentially expressed genes MuRF1, Atp2b2, and Elovl7 mRNA in the right ventricle
Fig. 3
Fig. 3
Differential gene expression of MuRF1 Tg + right ventricle after three weeks CH exposure. a. Summary of how differentially expressed genes in hypoxia-challenged MuRF1Tg + right ventricles were identified. b. Top 27 increased unique genes (> 3-fold) and top 18 decreased unique genes (<− 3-fold) compared to wild-typeMuRF1Tg+ RV tissue. Asterisk (*) designates transcript variant for duplicate gene name. MuRF1 Tg+: N = 3; strain-matched wild type: N = 3
Fig. 4
Fig. 4
Functional annotation of differentially expressed genes in MuRF1 Tg + right ventricles after three weeks CH exposure. a. Identification of ZIC1 protein binding sites based on binding sites in 5 of the differentially expressed genes in MuRF1 Tg + heart (Jakmip3, Cyp2b13, Gucd1, Kansl1l, Rab3gap1). The number of genes in dataset and enrichment (sites/sequence ratio) for each transcription factor is calculated to the right. b. STRING analysis of the top 27 genes increased (> 3-fold) and top 18 genes decreased (<− 3-fold) listed in Fig. 3b. c. Functional annotation of differentially expressed MuRF1 Tg + right ventricle by STRING analysis
Fig. 5
Fig. 5
Functional annotation of differentially expressed genes in MuRF1 Tg + hearts compared to wild-type controls. a. Hypercluster analysis of differentially expressed MuRF1−/− genes in the right ventricle after hypoxia challenge using the DAVID v6.8 Functional Annotation Bioinformatics database. Differentially expressed gene symbols are on the left, functional categories along the top (database source in parentheses). The cluster fold enrichment is color-coded (key in lower left). b. Reverse transcriptase quantitative PCR analysis of MuRF1−/− hearts of differentially expressed genes MuRF1, Casq1, and Myl1 mRNA in the right ventricle
Fig. 6
Fig. 6
Differentially expressed genes in a rat model of CH-induced pulmonary hypertension and right heart failure. a. Summary of how differentially expressed genes in CH-induced rat heart failure resulted in top 50 increased genes (> 4.9-fold) and top 50 decreased genes (< 3.9-fold) compared to normoxia using data from Drake, et al., 2013 [2]. Genes summarized in Additional file 4: Figure S2. b. Identification of transcription factors with binding sites of top 50 increased and top 50 decreased genes. The number of genes in the dataset and enrichment (sites/sequence ratio) for each transcription factor is calculated to the right
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