Cytoplasmic polyadenylation element binding protein 4 (Cpeb4) is a dynamic RNA-binding protein that regulates mRNA stability and translation by cytoplasmic polyadenylation. Cpeb4 has been identified to respond to hypertrophic stimuli in cardiomyocytes by changing its binding behaviour towards its target transcripts. Our published and unpublished data are demonstrating, that dysregulation of Cpeb4 contributes causally to heart failure (HF). However, its downstream targets remain partially understood.
To study the role of Cpeb4 in the heart, we conducted cardiomyocyte-specific overexpression (OE) using AAV9 mediated gene delivery in male and female mice. Cardiac remodelling was evaluated by Echocardiography, qPCR, Western Blot (WB) and Immunofluorescence Staining (IF). In vitro, gain and function approaches, as well as subcellular fractionation, apoptosis assays, WB and qPCR were performed in neonatal rat cardiomyocytes (NRCMs) for Cpeb4-dependent molecular and cellular effects.
Overexpression of Cpeb4 in vivo leads to dilated cardiomyopathy with reduced ejection fraction within 21 days of AAV9 induced OE, accompanied by an increased expression of the heart failure marker BNP. These findings were observed in both male and female mice. In NRCMs, Cpeb4 OE promoted apoptosis and an increased BNP expression, implying a function in cardiomyocyte survival and stress signalling. Subcellular fractionation demonstrated that Cpeb4 is not restricted to a single cellular compartment.
To unravel the mechanism by which the RNA binding protein Cpeb4 can cause such a drastic phenotype in cardiomyocytes, nanopore-sequencing and Ribo-seq was applied to identify transcripts regulated by Cpeb4. Ankrd1 was found to be regulated posttranscriptionally by Cpeb4. Ankrd1 is a cardiac stress-responsive protein involved in transcriptional and mechanosensitive signalling and has been implicated in several cardiac diseases. In Cpeb4 OE mouse hearts Ankrd1 was upregulated on the protein level without increases on the mRNA level, whereas in NRCMs and iPSC-derived cardiomyocytes Cpeb4 OE caused a downregulation of Ankrd1, suggesting a developmental stage-dependent regulation.
In summary, these data indicate a fundamental role of the RNA binding protein Cpeb4 in the regulation of pathological cardiac remodelling by its differential binding behaviour towards its target transcripts and demonstrates a pivotal interplay of translational and transcriptional regulation. These findings provide further evidence of the contribution of Cpeb4 dysregulation to the development of heart failure and highlight the importance of RNA binding proteins as regulators of cardiac diseases.
