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

Dasgupta T, Coram RJ, Stillwagon SJ, Ladd AN - PLoS ONE (2015)

Bottom Line: To the best of our knowledge, this was the first example of a genetically induced cardiomyopathy that spontaneously recovers without intervention.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Hopx and Fhl2 remain down-regulated in MHC-CELFΔ mice at 24 weeks.(A) We previously demonstrated that Hopx and Fhl2 transcripts and proteins are down-regulated in MHC-CELFΔ mice at 3 weeks [8]. 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 Srf transcripts were not significantly changed, Hopx and Fhl2 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).
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pone.0124462.g001: Hopx and Fhl2 remain down-regulated in MHC-CELFΔ mice at 24 weeks.(A) We previously demonstrated that Hopx and Fhl2 transcripts and proteins are down-regulated in MHC-CELFΔ mice at 3 weeks [8]. 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 Srf transcripts were not significantly changed, Hopx and Fhl2 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).

Mentions: We previously demonstrated that Hopx and Fhl2 transcripts and their corresponding proteins are reduced in MHC-CELFΔ mice compared to wild type littermates at 3 weeks [8]. This reduction in HOPX and FHL2 is accompanied by an increase in the expression of several SRF target genes despite no difference in SRF levels, presumably due to the alleviated inhibition of SRF activity (Fig 1A). We proposed that the combined effects of dysregulating the SRF transcriptional program and dysregulating the alternative splicing of CELF targets both contribute to cardiac dysfunction in MHC-CELFΔ mice [8]. In the mild line (MHC-CELFΔ-574), the state of the heart improves with age such that by 24 weeks the ejection fraction, fractional shortening, chamber dilation, wall thickness, and heart size are all comparable to wild type [16]. This recovery does not occur because splicing is restored to normal, however, as CELF targets continue to exhibit aberrant splicing patterns at 24 weeks [16]. To test whether normalization of the SRF program could be contributing to recovery in the mild line, we evaluated Srf, Hopx, and Fhl2 expression in transgenic females at 24 weeks of age (Fig 1B). As seen at 3 weeks, Hopx and Fhl2 transcripts are still significantly reduced in both mild and severe lines compared to wild type, while Srf remains at normal levels. SRF, HOPX, and FHL2 protein levels were also compared between transgenic and wild type animals by western blot (Fig 1C and 1D). As previously reported [8], HOPX and FHL2 levels were strongly reduced in MHC-CELFΔ mice at 3 weeks. Interestingly, HOPX levels were higher in transgenic hearts of the mild line at 24 weeks than at 3 weeks, although they remained lower than wild type. FHL2 levels also showed a trend towards increased levels at 24 weeks compared to 3 weeks, though this difference was not statistically significant. In contrast, HOPX and FHL2 levels did not change with age in either the wild type or severe line mice, where levels were higher to start with.


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

Dasgupta T, Coram RJ, Stillwagon SJ, Ladd AN - PLoS ONE (2015)

Hopx and Fhl2 remain down-regulated in MHC-CELFΔ mice at 24 weeks.(A) We previously demonstrated that Hopx and Fhl2 transcripts and proteins are down-regulated in MHC-CELFΔ mice at 3 weeks [8]. 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 Srf transcripts were not significantly changed, Hopx and Fhl2 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).
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pone.0124462.g001: Hopx and Fhl2 remain down-regulated in MHC-CELFΔ mice at 24 weeks.(A) We previously demonstrated that Hopx and Fhl2 transcripts and proteins are down-regulated in MHC-CELFΔ mice at 3 weeks [8]. 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 Srf transcripts were not significantly changed, Hopx and Fhl2 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).
Mentions: We previously demonstrated that Hopx and Fhl2 transcripts and their corresponding proteins are reduced in MHC-CELFΔ mice compared to wild type littermates at 3 weeks [8]. This reduction in HOPX and FHL2 is accompanied by an increase in the expression of several SRF target genes despite no difference in SRF levels, presumably due to the alleviated inhibition of SRF activity (Fig 1A). We proposed that the combined effects of dysregulating the SRF transcriptional program and dysregulating the alternative splicing of CELF targets both contribute to cardiac dysfunction in MHC-CELFΔ mice [8]. In the mild line (MHC-CELFΔ-574), the state of the heart improves with age such that by 24 weeks the ejection fraction, fractional shortening, chamber dilation, wall thickness, and heart size are all comparable to wild type [16]. This recovery does not occur because splicing is restored to normal, however, as CELF targets continue to exhibit aberrant splicing patterns at 24 weeks [16]. To test whether normalization of the SRF program could be contributing to recovery in the mild line, we evaluated Srf, Hopx, and Fhl2 expression in transgenic females at 24 weeks of age (Fig 1B). As seen at 3 weeks, Hopx and Fhl2 transcripts are still significantly reduced in both mild and severe lines compared to wild type, while Srf remains at normal levels. SRF, HOPX, and FHL2 protein levels were also compared between transgenic and wild type animals by western blot (Fig 1C and 1D). As previously reported [8], HOPX and FHL2 levels were strongly reduced in MHC-CELFΔ mice at 3 weeks. Interestingly, HOPX levels were higher in transgenic hearts of the mild line at 24 weeks than at 3 weeks, although they remained lower than wild type. FHL2 levels also showed a trend towards increased levels at 24 weeks compared to 3 weeks, though this difference was not statistically significant. In contrast, HOPX and FHL2 levels did not change with age in either the wild type or severe line mice, where levels were higher to start with.

Bottom Line: To the best of our knowledge, this was the first example of a genetically induced cardiomyopathy that spontaneously recovers without intervention.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus