Modulation of cytoplasmic polyadenylation element binding protein 3 changes electrophysiological features and calcium handling in cardiomyocytes

Background/Introduction

CPEB3 (Cytoplasmic Polyadenylation Element Binding Protein 3) is an RNA-binding protein capable of regulating mRNA translation. Previous studies have shown that downregulation of CPEB3 exacerbates myocardial infarction injury by inhibiting adaptive metabolic reprogramming, suggesting that CPEB3 may help maintain functional stability of ischemic myocardium. However, the role of CPEB3 in cardiomyocyte function has yet to be clarified.

Methods

Human induced pluripotent stem cells (hiPSCs) were generated from a healthy individual and subsequently differentiated into hiPSC-derived cardiomyocytes (hiPSC-CMs). The hiPSC-CMs were then transfected with pcDNA3.1-CPEB3 (for overexpression) and px459-shCPEB3 (for knockdown) vectors respectively. Transfection efficiency was assessed by Western blot, and the cells were subsequently used for further experiments. Patch-clamp analysis, Ca2+ imaging, single cell contraction, immunostaining and optical mapping analyses were conducted for the study.

Results

Overexpression of CPEB3 in hiPSC-CMs significantly prolonged action potential duration at 50% repolarization (APD50) and APD90, while resting membrane potential and action potential amplitude remained unchanged. Conversely, silencing CPEB3 induced opposite effects. Ionic current analysis indicated that the APD prolongation was primarily driven by an increased L-type Ca²⁺ current (ICa,L) density, with no significant alterations in Na⁺ or K⁺ currents, suggesting a Ca²⁺-driven electrophysiological regulatory mechanism.

Calcium transient analysis revealed that CPEB3 overexpression accelerated Ca²⁺ reuptake kinetics, reduced beat-to-beat variability, and enhanced Ca²⁺ cycling stability, whereas CPEB3 silencing impaired Ca²⁺ handling efficiency. Optical mapping of hiPSC-CM sheet showed that CPEB3 overexpression prolonged APD without affecting conduction properties.

Protein expression of CACNA1C, SERCA2a, and phosphorylated phospholamban (p-PLN) were increased by CPEB3 overexpression, whereas CPEB3 silencing reduced their expression.

Conclusion

This study demonstrates that modulation of CPEB3 induces changes in ion channel function and calcium handling related signaling in cardiomyocytes, suggesting potential roles of CPEB3 in electrophysiological and contractile function of heart.Optical mapping figure