Modulation of angiotensin II-mediated cardiac remodeling by the MEF2A target gene Xirp2

Abstract

Rationale: The vasoactive peptide angiotensin II (Ang II) is a potent cardiotoxic hormone whose actions have been well studied, yet questions remain pertaining to the downstream factors that mediate its effects in cardiomyocytes.

Objective: The in vivo role of the myocyte enhancer factor (MEF)2A target gene Xirp2 in Ang II-mediated cardiac remodeling was investigated.

Methods and results: Here we demonstrate that the MEF2A target gene Xirp2 (also known as cardiomyopathy associated gene 3 [CMYA3]) is an important effector of the Ang II signaling pathway in the heart. Xirp2 belongs to the evolutionarily conserved, muscle-specific, actin-binding Xin gene family and is significantly induced in the heart in response to systemic administration of Ang II. Initially, we characterized the Xirp2 promoter and demonstrate that Ang II activates Xirp2 expression by stimulating MEF2A transcriptional activity. To further characterize the role of Xirp2 downstream of Ang II signaling we generated mice harboring a hypomorphic allele of the Xirp2 gene that resulted in a marked reduction in its expression in the heart. In the absence of Ang II, adult Xirp2 hypomorphic mice displayed cardiac hypertrophy and increased beta myosin heavy chain expression. Strikingly, Xirp2 hypomorphic mice chronically infused with Ang II exhibited altered pathological cardiac remodeling including an attenuated hypertrophic response, as well as diminished fibrosis and apoptosis.

Conclusions: These findings reveal a novel MEF2A-Xirp2 pathway that functions downstream of Ang II signaling to modulate its pathological effects in the heart.

Figures

Figure 1. AngII regulates the Xirp2 promoter through MEF2A

(A) Xirp2 RT-PCR, 10µmol/L AngII-treated NRVMs (upper panel), 2 pooled samples each, GAPDH internal control (lower panel). (B) Xirp2 promoter constructs used in luciferase reporter assays. “M” denotes −75 MEF2 site, “X” denotes mutated −75 MEF2 site, sequences of wild type (M) and mutant (X) −75 MEF2 sites. (C) −1425 and −285 Xirp2 promoter constructs upon 10µmol/L AngII stimulation (2.5-fold, 2.4-fold respectively) vs. basal activity. −285 construct with AngII vs. −285mut with AngII (2.4-fold vs. 1.1-fold respectively, *p<0.05). (D) −1425 promoter co-transfected with MEF2A and AngII vs. MEF2A alone (10.6-fold vs. 3.4-fold respectively, #p<0.05). MEF2A/AngII activation of the −1425 promoter induced to similar levels in the −285 promoter construct (8.1-fold) but significantly reduced in the −1425mut (1.4-fold, *p<0.05), −285mut (2.6-fold, *p<0.05) and the −1425/−285 (2.5-fold, *p<0.05) promoters. Luciferase data represents triplicate wells, at least 3 experimental replicates. (C,D) Error bars represent ±1SEM. (E) RT-PCR analysis in NRVMs transduced with MEF2A-shRNA or LacZ-shRNA adenoviruses in presence/absence of 10µmol/L AngII treatment. “Xirp2-high” and “Xirp2-low” are the same gel imaged at 2 different exposures. MOI, multiplicity of infection.

Figure 2. Targeting strategy of Xirp2 gene and Xirp2 hypomorphic allele

(A) Conditional targeting of the Xirp2 gene. Genotyping primers for wt (a+c) and targeted loxP-neo (a+b). (B) RT-PCR on cardiac cDNA with primers (d+e). Normal transcript (exons 3–7) in wt (+/+), normal and truncated (exons 3,7) product in heterozygote (+/loxP), and truncated product in (loxP/loxP) mice. Sequencing truncated (exons 3,7) product reveals an in-frame splice. (C) Xirp2 qRT-PCR with primers spanning exons 2/3. loxP-neo/loxP-neo vs. wt expression in heart (19%, **p<0.005) and skeletal muscle (17%, *p<0.05), n=3 wt, n=9 loxP-neo/loxP-neo, B2M internal control, results representative of multiple experiments. (D) Xirp2 Western blot (>360kDa top band), hindlimb (left panel) and cardiac (right panel) muscle extracts, 20µg protein/lane, (*) indicates cross-reactivity with Xin isoforms described previously (11). (E) Xin qRT-PCR, loxP-neo/loxP-neo (Hypo) hearts vs. wt (n.s., p>0.05), n=3 wt, n=3 hypo, B2M internal control. (C, E) Error bars indicate ±1SEM.

Figure 3. Unstressed Xirp2 hypomorphic mice display cardiac hypertrophy

(A) H&E-stained heart sections (left panel) of wt and hypomorphic (hypo) mice. HW:BW (right panel), 19% increase in hypo vs. wt (ages 9–15 weeks) (**p<0.005), n=6 wt, n=14 hypo. Mean BW for wt and hypo: 22.4g and 22.3g, respectively. (B) Anti-vinculin immunostained ventricular myocytes (left panel). 1.6-fold increase in hypo CSA (*p<0.05), n=3 wt, n=3 hypo, 3 images/animal, ~100 myocytes/image. (A,B) Error bars indicate ±1SEM.

Figure 4. Global dysregulation of Xirp2 hypomorphic cardiac gene expression

(A) qRT-PCR analysis, hypo vs. wt ANF, BNP, and αMHC expression (n.s., p>0.05). Hypo vs. wt βMHC expression (4-fold, *p<0.005), n=3 to 8 per group. (B) Functional categories of genes dysregulated 2-fold or greater in microarray analysis (hypo vs. wt). (C) qRT-PCR of selected genes from microarray. Hypo vs. wt MARCKS (2.3-fold, **p<0.005), Pdlim3/ALP (2.8-fold, *p<0.05), Lipocalin2 (1.7-fold, **p<0.005), and RCAN1/MCIP1 (0.5-fold, *p<0.05). n=4 to 8 per group. (A,C) Data representative of multiple experiments, error bars indicate ±1SEM.

Figure 5. Attenuated response to AngII-induced cardiac remodeling in Xirp2 hypomorphic mice

(A) H&E staining (left panel). HW:BW (right panel), AngII-wt vs. sham-wt, 20% increase (**p<0.005), AngII-hypo vs. sham-hypo (n.s., p>0.05), n=4 sham-wt, n=3 sham hypo, n=8 AngII-wt, n=6 AngII-hypo. (B) Anti-vinculin immunostained cardiac sections (left panel). CSA quantification (right panel), AngII-wt vs. sham-wt CSA (2.7-fold,*p<0.05), AngII-hypo vs. sham-hypo (1.7-fold,**p<0.005), n=3 to 4 per group, 3 images/animal, ~100 cardiomyocytes/image. (C) Masson’s trichrome staining (left panel), arrows indicate fibrotic lesions. Quantification (right panel), AngII-wt vs. sham-wt (2.8--fold,*p<0.05), AngII-hypo vs. sham-hypo (n.s., p>0.05), AngII-wt vs. AngII-hypo (*p<0.05), n=3 to 5 per group, ~20 images/animal. (D) TUNEL-assay (left panel), arrows indicate apoptotic nuclei. Quantification (right panel), AngII-wt vs. sham-wt (3.3-fold,**p<0.005), AngII-hypo vs. sham-hypo (n.s., p>0.05), AngII-wt vs. AngII-hypo (*p<0.05), n=3 to 4 per group, ~100 images/animal. (A–D) Error bars indicate ±1SEM.

Figure 6. Expression of hypertrophic marker genes in AngII-infused hypomorphic mice

(A) qRT-PCR analysis, ANF expression in AngII-wt vs. wt (5.1-fold, *p<0.05) and in AngII-hypo vs. hypo (6.5-fold, *p<0.05), (n.s., p>0.05). BNP and αMHC expression (n.s., p>0.05). βMHC expression in AngII-wt vs. wt (5.7-fold, *p<0.05), AngII-hypo vs. hypo (n.s., p>0.05). B2M internal control, expression relative to untreated wt average, n=3 to 8 per group, results representative of multiple experiments. (B) Western blot, total and phospho-serine-9 (PS9)GSK-3β. Wt (pooled, n=3), hypo (pooled n=3), individual AngII-wt (n=4) and individual AngII-hypo (n=3) cardiac samples. Band-intensity quantified with Image-J, PS9-GSK-3β (upper blot) normalized to total GSK-3β (lower blot), AngII-hypo vs. AngII-wt (1.3-fold vs. 1.6-fold, *p<0.05). (C) Western blot, β-catenin levels in wt (pooled, n=3), hypo (pooled n=3), and individual AngII-wt (n=4) and AngII-hypo (n=3) cardiac samples. Band-intensity quantified with Image-J, β-catenin (upper blot) normalized to GAPDH (lower blot), AngII-hypo vs. AngII-wt (1.0-fold vs. 1.4-fold, *p<0.05). (B,C) Relative band intensity calculated relative to wt. (A–C) Error bars indicate ±1SEM.