The present gene expression profiling study implicates dysregulation of contractile gene expression programs as a primary cause of cardiomyopathy and cardiac dysfunction in MHC-CELFΔ mice. The dominant negative protein expressed in the hearts of these mice is restricted to the nucleus 
. Thus, dysregulation of CELF-mediated alternative splicing likely underlies these changes, although disruption of other nuclear functions of the CELF proteins (e.g., RNA editing 
) cannot be ruled out. Although this type of microarray does not reveal changes in alternative splicing, a few CELF targets identified to date encode proteins with known roles in contraction. For example, ankyrin 2 (ANK2) is required for targeting and stability of the Na+
exchanger 1 in cardiomyocytes 
. Loss-of-function has been implicated in inherited cardiac arrhythmia with increased risk for sudden cardiac death, likely due to elevation of calcium transients 
. Alternative splicing of Ank2
exon 21 was previously found to be regulated during heart development, and to respond to CELF1 over-expression 
alternative splicing is altered in the MHC-CELFΔ severe line, but not the mild line (Figure S4
Another striking finding of our current study is the dysregulation of the SRF transcription factor network in CELF-repressed and over-expressing mice. SRF is recognized for being at the convergence of multiple signaling pathways, known not only for controlling the transcription of immediate early response genes, such as Fos
, but also for the transcriptional activation of a large number of cardiac genes involved in contractile apparatus structure and function 
. Regulation by SRF is a common feature of many genes up-regulated during cardiac hypertrophy 
. Heart-specific over-expression of SRF in transgenic mice leads to the development of cardiac hypertrophy, chamber dilation, and impaired cardiac function 
, not very dissimilar from what we observe in MHC-CELFΔ mice. SRF levels normally increase slightly in the heart with age, and low levels of over-expression in the heart mimic cardiac aging, including myocardial cell hypertrophy and reduced cardiac function 
. Over-expression of SRF alone activates only a subset of its targets, however, and is not sufficient to induce the full hypertrophic gene expression profile in cardiomyocytes 
. SRF has low inherent transactivation activity by itself, but has strong transcriptional activity when working in concert with other transcription factors 
. Several other transcription factors that have been implicated in cardiac hypertrophy, including Nkx2.5 and HAND proteins 
, were not identified in the network analysis of our microarray data. A regulatory network for GATA4 was identified as dysregulated in the MHC-CELFΔ heart, but the handful of GATA4 targets affected exhibited a mixture of up- and down-regulation, together failing to suggest a consistent increase or decrease in GATA4 activity (data not shown).
CELF-mediated changes in SRF activity can likely be attributed to the levels of the inhibitory proteins, HOPX and FHL2, as the levels of SRF protein and alternative splicing of Srf
transcripts did not differ between wild type and transgenic animals ( and , and data not shown). Over-expression of CELF1 did lead to a reduction in Srf
transcript levels, but SRF protein levels were maintained, suggesting there may be a compensatory mechanism in place to maintain SRF levels in the myocardium. HOPX negatively regulates SRF activity by directly interacting with the SRF protein and inhibiting its binding to DNA 
. Ablation of HOPX is sufficient to induce an up-regulation of some SRF target genes, and can lead to cardiac hypertrophy in mice 
. FHL2 likewise inhibits transactivation of transcription by SRF via a direct protein:protein interaction 
. FHL2 knockout mice undergo normal cardiac development, but exhibit an exaggerated hypertrophic response following β-adrenergic stimulation 
. Both HOPX
are down-regulated in human heart failure 
, and may contribute to the dysregulation of SRF-dependent gene expression during pathogenesis. Not all of the SRF targets that were up-regulated in MHC-CELFΔ mice were down-regulated in MCKCUG-BP1 mice. Neither HOPX nor FHL2 completely repress SRF activity on all of its targets 
. Thus, the combination of HOPX and FHL2 down-regulation in the MHC-CELFΔ heart may have a greater stimulatory effect on SRF activity than the repressive effect of up-regulating FHL2 alone in MCKCUG-BP1 hearts. Congruent with this, Fos
is not repressed in the MCKCUG-BP1 heart, and Fos
expression has been reported to be unresponsive to FHL2 
. Strikingly, even though SRF plays a pivotal role in the maintenance of cardiac structure and function during development and pathogenesis, HOPX and FHL2 levels were not significantly affected in the two heart-specific SRSF1 and SRSF2 knockout mice 
. Taken together, the data from all of these mouse models indicate that the level of CELF activity plays a specific role in modulating the level of SRF activity in heart muscle via its interacting proteins.
Two SRF targets, Casq1
, were up-regulated in response to either CELF repression or CELF1 over-expression. Neither of these genes is up-regulated in several other mouse models of dilated cardiomyopathy or cardiac hypertrophy 
. This suggests that while these genes may respond specifically to dysregulation of CELF proteins, the modulation of SRF activity by HOPX and FHL2 is not the only determinant of their steady state levels.
The mechanism by which CELF-mediated alternative splicing regulates Hopx
levels is currently unknown. Alternative splicing can lead to changes in transcript and/or protein levels through the introduction or removal of regulatory elements in the untranslated regions that control transcript stability, localization, or translation 
. In addition, some transcript variants contain premature termination codons, which can target a transcript for degradation via the nonsense-mediated mRNA decay (NMD) pathway 
. We were unable to detect any change in Hopx
alternative splicing in MHC-CELFΔ hearts compared to wild type (data not shown), but a variant that is subject to NMD would by definition be rapidly decayed and potentially difficult to detect. It is also possible that the mechanism of Hopx
down-regulation is indirect, through the regulation of a factor that in turn modulates Hopx
levels. Little is currently known about the regulation of Hopx
expression in the heart. The elucidation of additional CELF targets could help establish this link.
The extent to which HOPX and FHL2 levels are down-regulated correlates with the development of cardiomyopathy in MHC-CELFΔ severe line males (). Surprisingly, however, HOPX and FHL2 are down-regulated to a greater extent in the mild line than the severe line ( and ). This may be the result of differences in integration site and/or genetic drift between the two lines. Alternatively, an additional compensatory mechanism may be activated that partially restores HOPX and FHL2 expression in the severe line, but not in the less dysfunctional mild line. In any case, the degree of de-repression of SRF alone is insufficient to explain differences in pathogenesis. Notably, the extent to which alternative splicing is disrupted also correlates with disease in the severe line males, as well as in females of the two lines (
and Figure S4
). Thus, it is likely the combined effects of dysregulation of SRF and alternative splicing that ultimately determine the extent to which contractile function in the heart is disrupted and dilated cardiomyopathy ensues.
Through the regulation of the SRF transcriptional network and the regulation of alternative splicing of cardiac transcripts, CELF proteins directly or indirectly exert control over cardiac gene expression at both transcriptional and post-transcriptional levels (). CELF-mediated alternative splicing programs in the heart may therefore represent an important regulatory node for modulating cardiac function during development, health, and disease.
CELF-mediated alternative splicing regulates contractile function via transcriptional and post-transcriptional control mechanisms.