Polyadenylation adds a poly(A) tail to the 3' end of eukaryotic mRNAs and thereby influences RNA stability, localization, and translation. Alternative polyadenylation (APA) generates transcript isoforms with distinct 3'UTRs and is increasingly recognized as an important layer of gene regulation in heart failure (HF). In failing DCM hearts, 80% of transcripts containing at least one cytoplasmic polyadenylation element (CPE), the canonical binding motif of Cytoplasmic Polyadenylation Element Binding Protein 4 (CPEB4), display 3'UTR lengthening due to APA, potentially increasing post-transcriptional control by RNA-binding proteins.
We identified CPEB4 as a dynamic RNA-binding protein that responds to hypertrophic stimulation in cardiomyocytes by changing its binding behavior toward target transcripts. In vivo gain- and loss-of-function experiments demonstrated that dysregulation of CPEB4 causally contributes to HF. In human ICM and DCM hearts, CPEB4 expression is increased, while its intron-retaining non-coding splice variant tends to be reduced, suggesting disease-associated isoform remodeling.
To define the underlying mechanisms, we performed a multi-omics approach combining direct mRNA Nanopore sequencing and Ribo-seq in AAV9-CPEB4-treated mice. Integration of these datasets showed that CPEB4 promotes poly(A) tail lengthening of transcripts containing a CPE. These transcripts were enriched for pathways related to cardiac muscle contraction, protein folding, and oxidative stress response. In parallel, Ribo-seq revealed altered translation of genes involved in mitochondrial function, extracellular matrix organization, and lipid metabolism, indicating that CPEB4 exerts broad post-transcriptional control over cardiac stress adaptation.
Among the regulated transcripts, we identified ZEB1 as a relevant CPEB4 target. ZEB1 is a transcriptional regulator of stress responses and essential for cardiac homeostasis. To define its functional role, we analyzed cardiomyocyte-specific and inducible Zeb1 knockout mouse models. Loss of Zeb1 caused cardiac dysfunction in both sexes, but females developed a markedly more severe dilated cardiomyopathy phenotype with early mortality, profound reduction in ejection fraction, ventricular dilation, hypertrophy, fibrosis, and increased expression of remodeling markers. Male mice showed a milder course, while inducible adult deletion caused systolic dysfunction predominantly in females. Ultrastructural analyses further revealed female-specific sarcomeric disarray.
Transcriptomic profiling uncovered pronounced sex-specific effects of Zeb1 deletion. While a core set of deregulated genes was shared between sexes, integration with ZEB1 ChIP-seq demonstrated distinct direct regulatory programs. In females, ZEB1 mainly repressed genes linked to epithelial proliferation and cell division, whereas in males it supported metabolic and mitochondrial gene networks, including pathways related to the TCA cycle and ATP synthesis.
Together, these findings identify a CPEB4-ZEB1 axis linking post-transcriptional poly(A) tail regulation to sex-specific transcriptional remodeling in HF and establish both factors as important regulators of cardiac integrity.
