Gene Expression Analyses during Spontaneous Reversal of Cardiomyopathy in Mice with Repressed Nuclear CUG-BP, Elav-Like Family (CELF) Activity in Heart Muscle

<div><p>CUG-BP, Elav-like family (CELF) proteins regulate cell type- and developmental stage-specific alternative splicing in the heart. Repression of CELF-mediated splicing activity via expression of a nuclear dominant negative CELF protein in heart muscle was previously shown to induce dysregulation of alternative splicing, cardiac dysfunction, cardiac hypertrophy, and dilated cardiomyopathy in MHC-CELFΔ transgenic mice. A “mild” line of MHC-CELFΔ mice that expresses a lower level of the dominant negative protein exhibits cardiac dysfunction and myopathy at a young age, but spontaneously recovers normal cardiac function and heart size with age despite the persistence of splicing defects. To the best of our knowledge, this was the first example of a genetically induced cardiomyopathy that spontaneously recovers without intervention. In this study, we explored the basis for this recovery. We examined whether a transcriptional program regulated by serum response factor (SRF) that is dysregulated in juvenile MHC-CELFΔ mice is restored in the mild line with age, and evaluated global changes in gene expression by microarray analyses. We found that differences in gene expression between the mild line and wild type hearts are greatly reduced in older animals, including a partial recovery of SRF target gene expression. We did not find evidence of a new compensatory pathway being activated in the mild line with age, and propose that recovery may occur due to developmental stage-specific compatibility of CELF-dependent splice variants with the cellular environment of the cardiomyocyte.</p></div

The number of affected genes in mild and severe lines at 3 and 24 weeks.

<p>Venn diagrams show the respective overlap of affected genes in MHC-CELFΔ-574 (“mild”) or MHC-CELFΔ-10 (“severe”) lines at 3 versus 24 weeks as compared to wild type mice at the same age.</p

The expression levels of transcripts identified by microarray as affected at 24 weeks, but not 3 weeks, in the mild line were evaluated by real-time RT-PCR.

<p>Total RNA was extracted and mRNA levels were assayed using SYBR green-based detection. <i>Myh7</i> was mildly reduced in both mild and severe lines at 3 weeks, but was restored to normal levels specifically in the mild line at 24 weeks. <i>Phlda1</i> was specifically up-regulated in the mild line at 24 weeks, but was elevated in both juvenile and mature mice of the severe line. <i>Amy2-2</i> levels went down with age in both lines, whereas <i>Sunc1</i> levels improved in the mild line and worsened in the severe line with age. Fold changes shown represent the mean + standard error of the mean of four to five independent samples for each group. An asterisk indicates a significant difference from wild type mice at the same age, and a pound sign indicates a significant difference from mice of the same group at 3 weeks (P ≤ 0.05).</p

Calcium handling genes are less disrupted at 24 weeks than at 3 weeks in the mild line.

<p>(A) Total RNA was extracted and mRNA levels were assayed by qRT-PCR using SYBR green-based detection. <i>Pln</i> and <i>Ryr2</i> levels were reduced in both lines of transgenic mice compared to wild type, but while <i>Pln</i> and <i>Ryr2</i> levels went up in the mild line with age, they went down further in the severe line. Fold changes shown represent the mean + standard error of the mean of five independent samples for each group. An asterisk indicates a significant difference from wild type mice at the same age, and a pound sign indicates a significant difference from mice of the same group at 3 weeks (P ≤ 0.05). (B) In heart muscle, myofilament relaxation and contraction are driven by calcium cycling. Relaxation occurs when Ca²⁺ levels in the cytoplasm drop following uptake into the sarcoplasmic reticulum (SR), an intracellular calcium storage site, via SR or endoplasmic reticulum Ca²⁺-ATPase 2a (SERCA2a). Phospholamban (PLN) restricts Ca²⁺ uptake by inhibiting SERCA2a. An influx of calcium into the cell activates release of Ca²⁺ from the SR into the cytosol via the ryanodine receptor (RyR2), triggering contraction. Down-regulation (indicated by green coloring) of PLN and RyR2 in the mild line at 3 weeks would lead to a depletion of Ca²⁺ from the cytoplasm by increasing SERCA2a-mediated uptake and reducing RyR2-mediated release. This would result in reduced contractile performance. At 24 weeks, partial restoration of PLN and RyR2 to closer to normal levels would restore normal calcium handling and improve contractility.</p

<i>Hopx</i> and <i>Fhl2</i> remain down-regulated in MHC-CELFΔ mice at 24 weeks.

<p>(A) We previously demonstrated that <i>Hopx</i> and <i>Fhl2</i> transcripts and proteins are down-regulated in MHC-CELFΔ mice at 3 weeks [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124462#pone.0124462.ref008" target="_blank">8</a>]. HOPX and FHL2 are known inhibitors of the cardiac transcription factor SRF, and reductions in HOPX and FHL2 are accompanied by increases in SRF target genes without a corresponding increase in SRF levels. (B) Total RNA was extracted from the hearts of 24 week-old wild type and MHC-CELFΔ mice, and mRNA levels were assayed by qRT-PCR using SYBR green-based detection. While <i>Srf</i> transcripts were not significantly changed, <i>Hopx</i> and <i>Fhl2</i> remain down-regulated in the hearts of both lines of MHC-CELFΔ mice at 24 weeks. Fold changes shown represent the mean + standard error of the mean of three independent sample sets. An asterisk indicates a significant difference from wild type (P ≤ 0.05). (C) Representative western blots of wild type, MHC-CELFΔ mild and severe line females at 3 and 24 weeks of age. Equivalent loading was further confirmed by Ponceau S staining (data not shown). (D) Quantitation of western blots (n = 3) shows an increase in HOPX expression in transgenic mice from the mild line at 24 weeks compared to 3 weeks, although levels still remain reduced compared to wild type hearts at the equivalent age. There is a trend towards increased FHL2 levels in the mild line at 24 weeks as well, but it is not statistically significant compared to the mild line at 3 weeks, and remains significantly lower than wild type. HOPX and FHL2 levels do not change over time in the severe line, and remain lower than wild type. SRF levels vary only slightly, and do not differ between wild type and the mild line at 3 or 24 weeks. An asterisk indicates a significant difference from wild type mice at the same age, and a pound sign indicates a significant difference from mice of the same group at 3 weeks (P ≤ 0.05).</p

SRF target genes are less dysregulated at 24 than 3 weeks in the mild line.

<p>(A) Representative western blots of wild type, MHC-CELFΔ mild and severe line females at 3 and 24 weeks of age. Equivalent loading was further confirmed by Ponceau S staining (data not shown). (B) Quantitation of western blots (n = 3) shows an increase in the SRF targets ACTA1 and FHL1 in wild type hearts at 24 weeks compared to 3 weeks. Although ACTA1 and FHL1 levels are elevated in both transgenic lines at 3 weeks, levels are close to those observed in wild type at 24 weeks. An asterisk indicates a significant difference from wild type mice at the same age, and a pound sign indicates a significant difference from mice of the same group at 3 weeks (P ≤ 0.05). (C) Total RNA was extracted and mRNA levels were assayed by qRT-PCR using SYBR green-based detection. While <i>Tpm2</i>, <i>Egr1</i>, <i>Rcan1</i>, and <i>Nppb</i> levels were all significantly up-regulated in hearts of MHC-CELFΔ-574 mice at 3 weeks [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124462#pone.0124462.ref008" target="_blank">8</a>], only <i>Rcan1</i> and <i>Nppb</i> remain up-regulated in the mild line at 24 weeks. All four SRF targets showed trends towards increased expression in MHC-CELFΔ-10 mice at 24 weeks, but did not reach statistical significance. Fold changes shown represent the mean + standard error of the mean of three independent sample sets. An asterisk indicates a significant difference from wild type (P ≤ 0.05).</p

Additional file 2: Movie S1. of Muscleblind-like 1 is required for normal heart valve development in vivo

Imaging of the AVC cushions in a representative wild type heart at E10.5 by OCT. Optical sections through a representative E10.5 wild type heart are shown. The AVC cushions are highlighted in purple, and a three-dimensional reconstruction of the AVC cushions is shown. A still image of this 3D reconstruction is shown in Fig. 3h, but has been rotated and scaled to match the orientation and magnification of the Mbnl1 ∆E3/∆E3 image in Fig. 3i. (MOV 2333 kb

Additional file 1: Figure S1. of Muscleblind-like 1 is required for normal heart valve development in vivo

MBNL1 expression in the embryonic brain. Immunohistochemistry with an anti-MBNL1 antibody was performed on sagittal sections from E9 wild type mouse embryos. Close-up of the head shows strong MBNL1 expression in the ectoderm lining the entrance to Rathke’s pouch (Rp), forebrain (FB), midbrain (MB), and hindbrain (HB) with little to no detectable staining within the interior encephalic tissues (highlighted by asterisks). A representative section from one of four embryos is shown. (PDF 1247 kb

Additional file 5: Movie S3. of Muscleblind-like 1 is required for normal heart valve development in vivo

Imaging of the pulmonary valve in a representative wild type heart at E19.5 by OCT. A three-dimensional reconstruction of a representative wild type E19.5 heart is shown with the pulmonary valve highlighted in purple, followed by optical sections through the heart. The pulmonary valve is indicated in purple, and a three-dimensional reconstruction of the valve is shown. A still image of this 3D reconstruction is shown in Fig. 5a. (MOV 3438 kb

Additional file 7: Figure S2. of Muscleblind-like 1 is required for normal heart valve development in vivo

Color and pulsed Doppler ultrasound revealed valve regurgitation and atrial communication in Mbnl1 E∆3/∆E3 mice. (A) Sequential color Doppler ultrasound images show regurgitation across the aortic valve of an Mbnl1 ∆E3/∆E3 mouse. Arrows indicate the direction of blood flow. LV = left ventricle, Ao = aorta, AV = aortic valve. Pulsed Doppler ultrasound revealed regurgitation across Mbnl1 ∆E3/∆E3 (B) pulmonary, (C) aortic, and (D) mitral valves. Arrows indicate regurgitation peaks. Atrial communication was observed in some Mbnl1 ∆E3/∆E3 mice by (E) color and (F) pulsed wave Doppler ultrasound. RA = right atrium, LA = left atrium, RV = right ventricle, LV = left ventricle. (PDF 8654 kb