Research ArticleCARDIAC ARRHYTHMIA

Pitx2 modulates a Tbx5-dependent gene regulatory network to maintain atrial rhythm

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Science Translational Medicine  31 Aug 2016:
Vol. 8, Issue 354, pp. 354ra115
DOI: 10.1126/scitranslmed.aaf4891
  • Fig. 1. Removal of Tbx5 from the adult mouse results in spontaneous, sustained AF.

    (A) Representative ambulatory telemetry ECG of R26CreERt2 control mice and Tbx5fl/fl;R26CreERt2 10 days after receiving TM. ECGs are representative of n = 24 Tbx5fl/fl;R26CreERt2 and n = 10 R26CreERt2 mice. (B and C) Signal-averaged ECG waveforms of ~1000 consecutive beats (B) and Poincaré plots of RR interval against the subsequent RR + 1 interval (C) in R26CreERt2 (n = 10) and Tbx5fl/fl;R26CreERt2 (n = 24) mice. P wave is present in control but absent in Tbx5fl/fl;R26CreERt2 mice (arrows). Number of mice with absent P wave and irregularly irregular heart rhythm described in A and B, in Tbx5fl/fl;R26CreERt2 versus R26CreERt2. P = 7.6 × 10−9 by two-tailed Fisher’s exact test. (D) Intracardiac atrial electrogram recordings and surface ECG in R26CreERt2 and Tbx5fl/fl;R26CreERt2 mice. A, atrial electrical signal; V, far field ventricular electrical signal. Data are representative of n = 6 Tbx5fl/fl;R26CreERt2 and n = 3 R26CreERt2 mice. Number of mice with irregular atrial electrogram in Tbx5fl/fl;R26CreERt2 versus R26CreERt2. P = 0.012 by two-tailed Fisher’s exact test. (E) Pulsed-wave Doppler across mitral valve alongside surface ECG. “A” wave is present in R26CreERt2 and absent in Tbx5fl/fl;R26CreERt2 recordings, indicative of a lack of coordinated atrial contraction. Data are representative of n = 6 Tbx5fl/fl;R26CreERt2 and n = 3 R26CreERt2 mice. Number of mice with absent “A” wave in Tbx5fl/fl;R26CreERt2 versus R26CreERt2. P = 0.012 by two-tailed Fisher’s exact test. (F) Representative atrial voltage activation maps from R26CreERt2 and Tbx5fl/fl;R26CreERt2 mice 7, 12, and 14 days after completion of TM treatment. Atrial activation maps demonstrate that conduction waves traverse the atria in ~12 ms at 7 days, ~20 ms at 12 days, and ~40 ms at 14 days after TM treatment. n = 2 for each of the three groups. SVC, superior vena cava; RA, right atrium; LA, left atrium; RV, right ventricle; LV, left ventricle. Trans-atrial conduction speed in Tbx5fl/fl;R26CreERt2 versus R26CreERt2. P = 0.02 by analysis of variance (ANOVA). (G) Atrial macro-reentrant pathways observed in Tbx5fl/fl;R26CreERt2 mice 14 days after TM treatment. Reentrant pathway travels right to left within the posterior atrial wall and left to right through the anterior atrial wall. Images are representative of n = 2 mice at 14 days.

  • Fig. 2. AP abnormalities in Tbx5fl/fl;R26CreERt2 atrial cardiomyocytes are mediated by disrupted calcium handling.

    (A) Representative AP from atrial cardiomyocytes isolated from R26CreERt2 and Tbx5fl/fl;R26CreERt2 mice and the corresponding properties of the APs. Resting membrane potential (RMP), AP amplitude (APA), and APD at 50% (APD50) and at 90% (APD90) repolarization. Data are means ± SEM (Tbx5fl/fl;R26CreERt2, n = 30 cardiomyocytes; R26CreERt2, n = 14 cardiomyocytes; n > 5 animals per group). P values were determined by two-tailed t test. (B) Representative abnormal depolarization events—EAD, DAD, and spontaneous phase 4 depolarization—observed in atrial cardiomyocytes. Total numbers of abnormal spontaneous events were recorded in R26CreERt2 (n = 14) and Tbx5fl/fl;R26CreERt2 (n = 30) atrial cardiomyocytes from n > 5 animals per group. Data are means ± SEM. P values were determined by one-tailed Fisher’s exact test. (C) Representative tracings of calcium release in isolated cardiomyocytes from R26CreERt2 and Tbx5fl/fl;R26CreERt2 mice. Calcium was imaged with Fluo-4 dye, and myocytes were paced at 1 Hz. Properties of calcium transient spikes, including decay constant (τ), time to 50% decay, and time to peak, were recorded across R26CreERt2 (n = 11) and Tbx5fl/fl;R26CreERt2 (n = 54) cardiomyocytes from n > 5 animals in each group. Data are means ± SEM. P values were determined by two-tailed t test. (D) Representative APs of atrial cardiomyocytes isolated from R26CreERt2 and Tbx5fl/fl;R26CreERt2 adult mutant mice treated with calcium-chelating agent BAPTA (5 mM). AP properties of these cardiomyocytes were determined, including RMP, APD50, and APD90. Data are means ± SD from Tbx5fl/fl;R26CreERt2 (n = 11) and R26CreERt2 (n = 7) cardiomyocytes across n > 3 animals per group. P values were determined by either two-tailed t test for continuous measurements or two-tailed Fisher’s exact test for count-based measurements (EADs, DADs, spontaneous depolarizations, and total abnormal depolarization events).

  • Fig. 3. TBX5-PITX2 gene regulatory network for atrial rhythm control.

    (A) Volcano plot of relative transcript expression from the left atria of Tbx5fl/fl;R26CreERt2 versus R26CreERt2 mice. All significantly misregulated genes (q < 0.05) are labeled blue, and all nonsignificant transcripts are in red. TBX5 and PITX2 shared targets labeled. (B) Heatmap of all significantly misregulated (q < 0.05) transcripts in left atria of Tbx5fl/fl;R26CreERt2 (n = 6) and R26CreERt2 (n = 4) mice. Cladogram shows clustering of biological replicates. (C) Relative gene expression by quantitative polymerase chain reaction (qPCR) of known AF ion channels from left atria. Data are means normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and relative to R26CreERt2 expression. Data are means ± SEM (n = 4 R26CreERt2 and n = 7 Tbx5fl/fl;R26CreERt2). Experiments were performed in technical triplicate. *P < 0.05 versus R26CreERt2 controls by two-tailed t test. See Table 1 for complete list of genes analyzed. (D) PITX2 genomic locus (hg19) aligned with ATAC-seq of iPS cell–derived cardiomyocytes and 4C with the PITX2c promoter as viewpoint. The region cloned for enhancer activity is boxed in red. Below, a smaller scale view of assayed enhancer showing ATAC-seq, Encyclopedia of DNA Elements (ENCODE) deoxyribonuclease (DNase) hypersensitivity (DHS) (darker to lighter gray showing relative strength of DNase signal, darker being stronger signal), and vertebrate conservation (Cons.). The TBX5-binding motif, which should occur by chance every ~1000 nucleotides, is labeled. (E) Fold enrichment after TBX5 chromatin immunoprecipitation (ChIP) by qPCR from a control locus and PITX2 enhancer in the human left atrial appendage. Data are means ± SEM (n = 3). (F and G) In vitro luciferase response assay of the candidate PITX2 regulatory element in human embryonic kidney (HEK) 293T cells cotransfected with TBX5 expression vector or HL-1 atrial cardiomyocytes. Wild-type (WT) enhancer, full mutant variant enhancer lacking the TBX5 binding motif mutant enhancer, and single-nucleotide polymorphism (SNP) rs1906595 variant enhancer are shown. Data are means ± SEM normalized to empty pGl4.23 vector with TBX5 expression (n = 4 WT enhancer, n = 4 empty, n = 3 T-box mutant, n = 4 SNP variant). (H) In vitro luciferase response assay of candidate TBX5/PITX2 co-regulated elements at Gja1, Ryr2, Dsp, Atp2a2, and Scn5a in HEK cells cotransfected with TBX5 or TBX5 and PITX2 or HL-1 cardiomyocytes and corresponding T-box mutant enhancers. Data are means ± SEM, normalized to blank vector with corresponding overexpression (n = 5 for Atp2a2 enhancer + TBX5 and Atp2a2 enhancer + TBX5/PITX2; n = 3 for all other groups). Experiments in (E) to (H) were performed in technical triplicate. P values in (E) to (H) were determined by two-tailed t test.

  • Fig. 4. Pitx2 haploinsufficiency rescues Tbx5 haploinsufficiency in mice.

    (A) Relative transcript expression by qPCR in the left atria from Pitx2 and Tbx5 heterozygotes and Tbx5;Pitx2 compound heterozygotes. Data are means ± SEM normalized to R26CreERt2 mice (set as 1) (n = 11 R26CreERt2; n = 15 Tbx5fl/+;R26CreERt2; n = 4 Pitx2fl/+;R26CreERt2; n = 8 Tbx5fl/+;Pitx2fl/+;R26CreERt2). *P < 0.05, two-tailed t test. Experiments were performed in technical triplicate. (B) Representative Poincaré plot of RR interval versus the subsequent RR + 1 interval (n = 7 R26CreERt2, n = 6 Tbx5fl/+;R26CreERt2, n = 9 Pitx2fl/+;R26CreERt2, and n = 7 Tbx5fl/+;Pitx2fl/+;R26CreERt2). (C) P-wave duration and PR interval calculated from ambulatory telemetry ECG recordings from mice in (B). P values were determined by two-tailed t test. Data are means ± SEM. (D) Representative AP recordings from atrial myocytes isolated from R26CreERt2, Tbx5fl/+;R26CreERt2, Pitx2fl/+;R26CreERt2, and Tbx5fl/+;Pitx2fl/+;R26CreERt2 mice. RMP, APA, APD50, and APD90 repolarization were determined from n > 3 animals per group (n = 14 R26CreERt2 cells, n = 15 Tbx5fl/+;R26CreERt2, n = 14 Pitx2fl/+;R26CreERt2, and n = 13 Tbx5fl/+;Pitx2fl/+;R26CreERt2). P values were determined versus R26CreERT2 controls. Inappropriate depolarization P values were measured by two-tailed Fisher’s exact test; APD90 and APD50 were determined by two-tailed t test. (E) Pacing induction by intra-atrial pacing of R26CreERt2 (n = 10), Tbx5fl/+;R26CreERt2 (n = 7), Pitx2fl/+;R26CreERt2 (n = 5), and Tbx5fl/+;Pitx2fl/+;R26CreERt2 (n = 7) mice. P values were determined by two-tailed Fisher’s exact test.

  • Fig. 5.

    A TBX5-PITX2 regulatory loop regulates atrial rhythm. (A) Loss of TBX5 in the adult atrium leads to slowed atrial conduction, prolonged AP, and disrupted calcium handling, leading to AF trigger and substrate. (B) A TBX5-PITX2 incoherent feed-forward loop regulates atrial conduction genes Scn5a, Gja1, Ryr2, Atp2a2, and Dsp. TBX5 drives PITX2 expression. TBX5 and PITX2 positively and negatively regulate downstream targets. Misregulation of this loop disrupts atrial conduction.

  • Table 1. Gene expression changes in Tbx5fl/fl;R26CreERt2 mouse left atria.

    Gene expression of ion channels implicated in AF and of Tbx5/Pitx2 cotargets in left atrial tissue from Tbx5f/f;R26CreERt2 adult mutant mice normalized to R26CreERt2 by RNA-seq or qRT-PCR, in independent cohorts. Data are means ± SEM (n = 4 R26CreERt2 and n = 6 Tbx5f/f;R26CreERt2 for RNA-seq; n = 4 R26CreERt2 and n = 7 Tbx5fl/fl;R26CreERt2 for qRT-PCR). Nonsignificant changes in the RNA-seq were not validated by real time, with the exception of Pitx2c, which was just above significance by RNA-seq.

    GeneTbx5fl/fl;R26CreERt2 versus R26CreERt2 fold
    change by RNA-seq
    q
    (RNA-seq)
    Tbx5f/f;R26CreERt2 versus R26CreERt2 fold
    change by qRT-PCR
    P (qRT-PCR)
    Pitx2c0.530.070.22 ± 0.090.03
    Gja50.053 × 10−40.23 ± 0.052.49 × 10−6
    Gja10.243 × 10−40.30 ± 0.041.06 × 10−5
    Dsp0.363 × 10−40.44 ± 0.051.99 × 10−8
    Scn5a0.303 × 10−40.34 ± 0.071.06 × 10−5
    Ryr20.223 × 10−40.42 ± 0.072.14 × 10−6
    Atp2a20.423 × 10−40.42 ± 0.170.002
    Sln0.413 × 10−40.52 ± 0.051.2 × 10−4
    Kcnd30.677 × 10−30.25 ± 0.170.002
    Kcnj30.143 × 10−40.12 ± 0.019.56 × 10−12
    Kcnn20.770.100.33 ± 0.084.83 × 10−5
    Kcnj80.677 × 10−30.38 ± 0.043 × 10−6
    Kcna50.780.040.42 ± 0.134 × 10−4
    Kcnj20.603 × 10−40.42 ± 0.091.4 × 10−4
    Kcnj50.213 × 10−40.49 ± 0.24.7 × 10−5
    Abcc90.583 × 10−40.63 ± 0.041.7 × 10−5
    Cacna1c0.950.88
    Camk2a/b/d/g0.24/0.83/0.59/1.250.11/1/0.2/1
    Slc8a1/2/30.81/3.08/0.610.18/1/1
    Calm2/31.27/1.440.03/4 × 10−4
  • Table 2. Gene expression changes in Tbx5fl/+;R26CreERt2, Pitx2fl/+;R26CreERt2 , and Tbx5fl/+;Pitx2fl/+; R26CreERt2 mouse left atria.

    Gene expression TBX5/PITX2 shared targets in left atrial tissue normalized to R26CreERt2 by qRT-PCR, in independent cohorts. Data are means ± SEM (n = 4 R26CreERt2 and n = 6 Tbx5f/f;R26CreERt2 for RNA-seq; n = 4 R26CreERt2 and n = 7 Tbx5fl/fl;R26CreERt2 for qRT-PCR). *P < 0.05 versus R26CreERt2, P < 0.05 versus Tbx5fl/+;R26CreERt2. n = 11 R26CreERt2; n = 15 Tbx5fl/+;R26CreERt2; n = 4 Pitx2fl/+;R26CreERt2; n = 8 Tbx5fl/+;Pitx2fl/+;R26CreERt2. For Tbx5fl/+;R26CreERt2 versus R26CreERt2 Tbx5: P = 1.5 × 10−5; Pitx2c: P = 0.86; Scn5a: P = 0.028; Ryr2: P = 3.98 × 10 −5; Gja1: P = 6.98 × 10−3 and Atp2a2: P = 5.5 × 10−3. For Pitx2fl/+;R26CreERt2 versus R26CreERt2 Pitx2c: P = 0.05; Scn5a: P = 0.03; Tbx5: P = 0.47; Ryr2: P = 0.44; Gja1: P = 0.52; Atp2a2: P = 0.60. For Tbx5fl/+;Pitx2fl/+;R26CreERt2 versus Tbx5fl/+;R26CreERt2 Tbx5: P = 0.56; Scn5a: P = 7.5 × 10−3; Ryr2: P = 0.01; Gja1: P = 0.02; Atp2a2: P = 0.19; Dsp: P = 0.05. Tbx5fl/+;Pitx2fl/+;R26CreERt2 versus R26CreERt2 Scn5a: P = 0.58; Ryr2: P = 0.07; Gja1: P = 0.99; Atp2a2: P = 0.19; Dsp: P = 0.31. By two-tailed t test. Experiments performed in technical triplicate.

    Tbx5f/+;R26CreERt2
    fold change
    Pitx2f/+;R26CreERt2
    fold change
    Tbx5f/+;Pitx2fl/+;R26CreERt2
    fold change
    Tbx50.55 ± 0.06*1.11 ± 0.170.64 ± 0.05*
    Pitx2c0.91 ± 0.130.45 ± 0.10*0.54 ± 0.22*
    Ryr20.59 ± 0.04*1.12 ± 0.150.72 ± 0.14*
    Atp2a20.73 ± 0.05*1.10 ± 0.110.86 ± 0.08
    Dsp1.07 ± 0.070.88 ± 0.141.31 ± 0.21
    Gja10.70 ± 0.04*0.95 ± 0.060.98 ± 0.03
    Scn5a0.85 ± 0.08*1.95 ± 0.10*1.18 ± 0.23

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/354/354ra115/DC1

    Materials and Methods

    Fig. S1. No change in cardiac function after onset of AF in Tbx5fl/fl;R26CreERt2 mice.

    Fig. S2. Tbx5fl/fl;R26CreERt2 develop more severe ventricular arrhythmias over time.

    Fig. S3. Tbx5fl/fl;R26CreERt2 show paroxysmal AF.

    Fig. S4. Optical AP from right atrium of Tbx5fl/fl;R26CreERt2 mice.

    Fig. S5. Atrial rhythm instability and AF inducibility in Tbx5fl/+;R26CreERt2 is rescued by Pitx2 haploinsufficiency.

    Table S1. qRT-PCR primers used.

    Movie S1. Ex vivo optical mapping in Tbx5fl/fl;R26CreERt2 mice at 7 days after TM.

    Movie S2. Ex vivo optical mapping in Tbx5fl/fl;R26CreERt2 mice at 12 days after TM.

    Movie S3. Ex vivo optical mapping in Tbx5fl/fl;R26CreERt2 mice at 14 days after TM.

    References (3339)

  • Supplementary Material for:

    Pitx2 modulates a Tbx5-dependent gene regulatory network to maintain atrial rhythm

    Rangarajan D. Nadadur, Michael T. Broman, Bastiaan Boukens, Stefan R. Mazurek, Xinan Yang, Malou van den Boogaard, Jenna Bekeny, Margaret Gadek, Tarsha Ward, Min Zhang, Yun Qiao, James F. Martin, Christine E. Seidman, Jon Seidman, Vincent Christoffels, Igor R. Efimov, Elizabeth M. McNally, Christopher R. Weber, Ivan P. Moskowitz*

    *Corresponding author. Email: imoskowitz{at}uchicago.edu

    Published 31 August 2016, Sci. Transl. Med. 8, 354ra115 (2016)
    DOI: 10.1126/scitranslmed.aaf4891

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. No change in cardiac function after onset of AF in Tbx5fl/fl;R26CreERt2 mice.
    • Fig. S2. Tbx5fl/fl;R26CreERt2 develop more severe ventricular arrhythmias over time.
    • Fig. S3. Tbx5fl/fl;R26CreERt2 show paroxysmal AF.
    • Fig. S4. Optical AP from right atrium of Tbx5fl/fl;R26CreERt2 mice.
    • Fig. S5. Atrial rhythm instability and AF inducibility in Tbx5fl/+;R26CreERt2 is rescued by Pitx2 haploinsufficiency.
    • Table S1. qRT-PCR primers used.
    • Legends for movies S1 to S3
    • References (3339)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). Ex vivo optical mapping in Tbx5fl/fl;R26CreERt2 mice at 7 days after TM.
    • Movie S2 (.avi format). Ex vivo optical mapping in Tbx5fl/fl;R26CreERt2 mice at 12 days after TM.
    • Movie S3 (.avi format). Ex vivo optical mapping in Tbx5fl/fl;R26CreERt2 mice at 14 days after TM.

    [Download Movies S1 to S3]

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