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Impaired sarcoplasmic reticulum (SR) Ca2+ cycling and depressed contractility, a hallmark of human and experimental heart failure, has been partially attributed to increased protein phosphatase 1 (PP-1) activity, associated with down-regulation of its endogenous inhibitor-1. The levels and activity of inhibitor-1 are reduced in failing hearts, contributing to dephosphorylation and inactivation of key calcium cycling proteins. Therefore, we investigated the mechanisms that mediate decreases in inhibitor-1 by post-transcriptional modification.Bioinformatics revealed that 17 human microRNAs may serve as modulators of inhibitor-1. However, real-time PCR analysis identified only one of these microRNAs, miR-765, as being increased in human failing hearts concomitant with decreased inhibitor-1 levels. Expression of miR-765 in HEK293 cells or mouse ventricular myocytes confirmed suppression of inhibitor-1 levels through binding of this miR-765 to the 3'-untranslated region of inhibitor-1 mRNA. To determine the functional significance of miR-765 in Ca2+ cycling, pri-miR-765 as well as a non-translated nucleotide sequence (miR-Ctrl) were expressed in adult mouse ventricular myocytes. The inhibitor-1 expression levels were decreased, accompanied by enhanced PP-1 activity in the miR-765 cardiomyocytes, and these reflected depressed contractile mechanics and Ca2+ transients, compared with the miR-Ctrl group. The depressive effects were associated with decreases in the phosphorylation of phospholamban and SR Ca2+ load. These miR-765 negative inotropic effects were abrogated in inhibitor-1-deficient cardiomyocytes, suggesting its apparent specificity for inhibitor-1.miR-765 levels are increased in human failing hearts. Such increases may contribute to depressed cardiac function through reduced inhibitor-1 expression and enhanced PP-1 activity, associated with reduced SR Ca2+ load.