<p>Phospholamban (PLN) is a regulatory protein of the SERCA2α calcium transporter, which plays an important role in maintaining calcium homeostasis in cardiomyocytes. Deletion of the 14th arginine of PLN (PLN-R14del) leads to dysregulation of SERCA2α and PLN aggregation, and is a common cause of dilated cardiomyopathy. In this study, by using CRISPR-Cas9 gene editing technology, we constructed the PLN-R14del mouse model and hESCs. The PLN^{R14del/R14del} mice developed severe ventricular dilation, cardiac fibrosis, and PLN aggregation, as well as premature death due to heart failure. Reduced cardiomyocyte functions and PLN aggregation were also observed in the human PLN^R14del/WT cardiomyocytes differentiated from gene-edited hESCs. AAV delivery of PLN-L31A/I40A, which blocks PLN-R14del and SERCA2α interaction but without blocking the function of the latter, provided a therapeutic effect in both mice and human cardiomyocytes. These results not only suggest that PLN-L31A/I40A gene therapy is practical, but also suggest that blocking the interaction between PLN-R14del and SERCA2α with other modalities, such as small molecules, might also be beneficial.</p>