miR ‐221 Mediates the Regulation of Phospholamban Expression and Cardiac Contractility by ZBTB20

AIMS: Phospholamban (PLN) is a key regulator of sarco-endoplasmic reticulum calcium ATPase (SERCA) activity and myocardial contractility, but its expression control remains incompletely understood. This study seeks to clarify the molecular mechanism of PLN regulation and its functional relevance in cardiac physiology. METHODS AND RESULTS: Using cardiomyocyte-specific ZBTB20 knockout (CZB20KO) mice and primary cardiomyocytes, we demonstrated that ZBTB20 deficiency significantly reduced PLN protein at a higher magnitude than its mRNA levels, accompanied by a marked increase in the expression levels of miR-221 and miR-222 derived from the same gene cluster. Cardiomyocyte-specific deletion of miR-221/222 alone did not affect cardiac PLN expression in the mice, but fully restored the expression of PLN protein and the basal cardiac contractility in the context of ZBTB20 deficiency, as evidenced by normal left ventricular ejection fraction and fractional shortening compared to control mice. Through luciferase reporter assays with 3'UTR binding site mutagenesis and gain/loss-of-function experiments, we identified miR-221 but not its paralog miR-222 as a direct regulator of PLN by targeting its 3'UTR of mRNA. Moreover, cardiomyocyte-specific overexpression of miR-221 in mice led to a reduction in cardiac PLN protein levels. ChIP assay did not reveal significant binding of ZBTB20 to the miR-221/222 gene cluster. CONCLUSIONS: Our study identifies miR-221 as a novel regulator of cardiac PLN expression and a mediator of the regulation of PLN by ZBTB20. Thus this work provides insights into the regulation of basal contractility and functional reserve of the heart.