Up-regulation of micro-RNA765 in human failing hearts is associated with post-transcriptional regulation of protein phosphatase inhibitor-1 and depressed contractility

Abstract

Aims: Impaired sarcoplasmic reticulum (SR) Ca(2+) 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.

Methods and results: 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 Ca(2+) 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 Ca(2+) transients, compared with the miR-Ctrl group. The depressive effects were associated with decreases in the phosphorylation of phospholamban and SR Ca(2+) load. These miR-765 negative inotropic effects were abrogated in inhibitor-1-deficient cardiomyocytes, suggesting its apparent specificity for inhibitor-1.

Conclusions: 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 Ca(2+) load.

Keywords: Calcium cycling; Cardiomyocyte contractility; Heart failure; Inhibitor-1; MicroRNA-765; Protein phosphatase 1.

Figure 1

Expression levels of inhibitor-1 mRNA and its potential regulatory microRNAs in human hearts. Total mRNA was isolated and purified from human failing hearts (HFrEF) and non-failing hearts. (A) Representative real-time PCR curves illustrating inhibitor-1 and glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA expression in non-failing and failing heart samples. (B) Quantitative analysis of inhibitor-1 expression levels relative to the non-failing heart group after normalization to GAPDH levels. (C) Heat map illustrating expression of candidate microRNAs regulating inhibitor-1 in non-failing and failing hearts. (D) Representative real-time PCR curves illustrating hsa-miR-765 and U6 mRNA expression in non-failing and failing heart samples. (E) Quantitative analysis of hsa-miR-765 expression levels relative to the non-failing group after normalization to U6 levels. (n=7 hearts per group; ^*P <0.05 vs. the non-failing group).

Figure 2

Luciferase assays of the microRNA-765 (miR-765)-binding site on the human, mouse, and rat inhibitor-1 3′-untranslated region (UTR). (A–C) Predicted miR-765-binding sequences on the 3′-UTRs of human (A), mouse (B), and rat (C) inhibitor-1 mRNAs. (D–F) Luciferase activity analysis indicated that human (D), mouse (E), and rat (F) inhibitor-1 are authentic target of miR-765 (n=5–6 preparations per group; ^*P <0.05)/

Figure 3

Expression of human microRNA-765 (miR-765) in mouse cardiomyocytes. (A) Schematic diagram of the recombinant adenoviral vector. The primary human miR-765 gene as well as a scrambled DNA sequence were amplified by PCR and incorporated into CMVP (cytomegalovirus promoter) downstream in the Adeasy-1/shuttle backbone vector. (B) Representative immunoblots illustrating inhibitor-1, protein phosphatase-1 (PP-1), and glyceraldehyde phosphate dehydrogenase (GAPDH) in mouse cardiomyocytes. (C) Quantitative results of total inhibitor-1 mRNA and protein expression levels in mouse cardiomyocytes. (D) PP-1 activity was assessed in mouse cardiomyocytes. (E) Representative immunoblots illustrate the phosphorylation of phospholamban (PLN) and ryanodine receptor 2 (RyR2) in mouse cardiomyocytes. (F) Quantitative analysis of the phosphorylation levels of PLN at Ser16 and Thr17, normalized to their respective total PLN protein levels. (G) Quantitative analysis of the phosphorylation levels of RyR2 at Ser2809 and Ser2815, normalized to their respective total RyR2 protein levels (n=5–6 hearts/group per group; ^*P <0.05 vs. Admir-Ctrl).

Figure 4

Contractile parameters of isolated adult mouse cardiomyocytes in the presence of microRNA-765 (miR-765). Isolated mouse left ventricular myocytes were suspended in 1.0 mmol/L Ca²⁺ Tyrode solution and field stimulated at 0.5Hz. (A) Representative cell-shortening traces of cardiomyocytes treated with AdmiR-Ctrl or AdmiR-765. Fractional shortening (FS%) (B), rates of shortening (+dL/dt), (C) and rates of relaxation (−dL/dt) (D) under resting conditions (Basal) and in the presence of 100 nM isoproterenol. (Basal, AdmiR-Ctrl=87 cells from six hearts; AdmiR-765=103 cells from 6 hearts; isoproterenol, AdmiR-Ctrl=45 cells from five hearts; AdmiR-765=50 cells from five hearts; a minimum of nine cells were used/heart) (^*P <0.05)

Figure 5

Effects of microRNA-765 (miR-765) on cardiomyocytes calcium transients under basal and isoproterenol-stimulated conditions. Intracellular calcium transients were assessed by Fura2, a ratio metric fluorescent dye, which binds to free intracellular calcium. (A) Representative tracings of Ca²⁺ transients under basal conditions and in the presence of isoproterenol; Ca²⁺ transient peak (B); and time constant of the calcium transient decay (Tau) (C). (D) Representative curves for caffeine-induced calcium release fromthe sarcoplasmic reticulum (SR) under basal condition. (E) Peak caffeine-induced calcium release from the SR (SR load) is indicated by the Fura2 ratio of 340/380 nm, and the prolongation of time to 50% baseline is indicated by T50. (Basal, AdmiR-Ctrl=56 cells from six hearts; AdmiR-765=50 cells from six hearts; isoproterenol, AdmiR-Ctrl=50 cells from five hearts; AdmiR-765=48 cells from five hearts; a minimum of seven cells were used per heart) (^*P <0.05).

Figure 6

Effects of microRNA-765 (miR-765) overexpression on contractile function in inhibitor-1-deficient mouse cardiomyocytes. (A) Representative tracings for contraction in wild-type (WT) and inhibitor-1 knockout (I-1 KO) mouse cardiomyocytes post-AdmiR-Ctrl or AdmiR-765 infection. (B–D) Quantitative analysis of fractional shortening (FS%) (B), rates of shortening (+dL/dt) (C), and rates of relaxation (−dL/dt) (D). (E) Proposed mechanism of miR-765 regulation of cardiomyocyte contractility (WT infected with AdmiR-Ctrl=44 cells from four hearts; WT infected with AdmiR-765=53 cells from four hearts; I-1 KO infected with AdmiR-Ctrl=40 cells from four hearts; I-1 KO infected with AdmiR-Ctrl=47 cells from four hearts; a minimum of eight cells was used per heart) (^*P <0.05).