Sarah E. Sansom

MicroRNA-1 and -133 increase arrhythmogenesis in heart failure by dissociating phosphatase activity from RyR2 complex.

In heart failure (HF), arrhythmogenic spontaneous sarcoplasmic reticulum (SR) Ca2+ release and afterdepolarizations in cardiac myocytes have been linked to abnormally high activity of ryanodine receptors (RyR2s) associated with enhanced phosphorylation of the channel. However, the specific molecular mechanisms underlying RyR2 hyperphosphorylation in HF remain poorly understood. The objective of the current study was to test the hypothesis that the enhanced expression of muscle-specific microRNAs (miRNAs) underlies the HF-related alterations in RyR2 phosphorylation in ventricular myocytes by targeting phosphatase activity localized to the RyR2. We studied hearts isolated from canines with chronic HF exhibiting increased left ventricular (LV) dimensions and decreased LV contractility. qRT-PCR revealed that the levels of miR-1 and miR-133, the most abundant muscle-specific miRNAs, were significantly increased in HF myocytes compared with controls (2- and 1.6-fold, respectively). Western blot analyses demonstrated that expression levels of the protein phosphatase 2A (PP2A) catalytic and regulatory subunits, which are putative targets of miR-133 and miR-1, were decreased in HF cells. PP2A catalytic subunit mRNAs were validated as targets of miR-133 by using luciferase reporter assays. Pharmacological inhibition of phosphatase activity increased the frequency of diastolic Ca2+ waves and afterdepolarizations in control myocytes. The decreased PP2A activity observed in HF was accompanied by enhanced Ca2+/calmodulin-dependent protein kinase (CaMKII)-mediated phosphorylation of RyR2 at sites Ser-2814 and Ser-2030 and increased frequency of diastolic Ca2+ waves and afterdepolarizations in HF myocytes compared with controls. In HF myocytes, CaMKII inhibitory peptide normalized the frequency of pro-arrhythmic spontaneous diastolic Ca2+ waves. These findings suggest that altered levels of major muscle-specific miRNAs contribute to abnormal RyR2 function in HF by depressing phosphatase activity localized to the channel, which in turn, leads to the excessive phosphorylation of RyR2s, abnormal Ca2+ cycling, and increased propensity to arrhythmogenesis.

Chromosome 21-derived microRNAs provide an etiological basis for aberrant protein expression in human down syndrome brains.

Down syndrome (DS), or Trisomy 21, is the most common genetic cause of cognitive impairment and congenital heart defects in the human population. Bioinformatic annotation has established that human chromosome 21 (Hsa21) harbors five microRNA (miRNAs) genes: miR-99a, let-7c, miR-125b-2, miR-155, and miR-802. Our laboratory recently demonstrated that Hsa21-derived miRNAs are overexpressed in DS brain and heart specimens. The aim of this study was to identify important Hsa21-derived miRNA/mRNA target pairs that may play a role, in part, in mediating the DS phenotype. We demonstrate by luciferase/target mRNA 3′-untranslated region reporter assays, and gain- and loss-of-function experiments that miR-155 and -802 can regulate the expression of the predicted mRNA target, the methyl-CpG-binding protein (MeCP2). We also demonstrate that MeCP2 is underexpressed in DS brain specimens isolated from either humans or mice. We further demonstrate that, as a consequence of attenuated MeCP2 expression, transcriptionally activated and silenced MeCP2 target genes, CREB1/Creb1 and MEF2C/Mef2c, are also aberrantly expressed in these DS brain specimens. Finally, in vivo silencing of endogenous miR-155 or -802, by antagomir intra-ventricular injection, resulted in the normalization of MeCP2 and MeCP2 target gene expression. Taken together, these results suggest that improper repression of MeCP2, secondary to trisomic overexpression of Hsa21-derived miRNAs, may contribute, in part, to the abnormalities in the neurochemistry observed in the brains of DS individuals. Finally these results suggest that selective inactivation of Hsa21-derived miRNAs may provide a novel therapeutic tool in the treatment of DS.

Trisomy-21 gene dosage over-expression of miRNAs results in the haploinsufficiency of specific target proteins

Down syndrome (DS) or Trisomy 21 (Ts21) is caused by the presence of an extra copy of all or part of human chromosome 21 (Hsa21) and is the most frequent survivable congenital chromosomal abnormality. Bioinformatic annotation has established that Hsa21 harbors more than 400 genes, including five microRNA (miRNA) genes (miR-99a, let-7c, miR-125b-2, miR-155, and miR-802). MiRNAs are endogenous, single-stranded, small non-coding RNA molecules that regulate gene expression by interacting with specific recognition elements harbored within the 3′-untranslated region (3′-UTR) of mRNAs and subsequently target these mRNAs for translational repression or destabilization. MiRNA expression profiling, miRNA RT-PCR, and miRNA in situ hybridization experiments have demonstrated that Hsa21-derived miRNAs were over-expressed in fetal brain and heart specimens isolated from individuals with DS. We now propose that Ts21 gene dosage over-expression of Hsa21-derived miRNAs in DS individuals result in the decreased expression of specific target proteins (i.e. haploinsufficiency) that contribute, in part, to the DS phenotype.

Cardiovascular Disease, Single Nucleotide Polymorphisms; and the Renin Angiotensin System: Is There a MicroRNA Connection?

Essential hypertension is a complex disorder, caused by the interplay between many genetic variants, gene-gene interactions, and environmental factors. Given that the renin-angiotensin system (RAS) plays an important role in blood pressure (BP) control, cardiovascular regulation, and cardiovascular remodeling, special attention has been devoted to the investigation of single-nucleotide polymorphisms (SNP) harbored in RAS genes that may be associated with hypertension and cardiovascular disease. MicroRNAs (miRNAs) are a family of small, ∼21-nucleotide long, and nonprotein-coding RNAs that recognize target mRNAs through partial complementary elements in the 3′-untranslated region (3′-UTR) of mRNAs and inhibit gene expression by targeting mRNAs for translational repression or destabilization. Since miRNA SNPs (miRSNPs) can create, destroy, or modify miRNA binding sites, this review focuses on the hypothesis that transcribed target SNPs harbored in RAS mRNAs, that alter miRNA gene regulation and consequently protein expression, may contribute to cardiovascular disease susceptibility.

Increased Levels of Micrornas miR-1 and miR-133 in Failing Heart Underlie Dissociation of Phosphatase Activity from RyR2 Complex Resulting in Enhanced RyR2 CaMKII-Dependent Phosphorylation and Cardiac Arrhythmias

Increased propensity of ventricular myocytes to arrhythmogenic spontaneous SR Ca release and afterdepolarizations in heart failure (HF) has been linked to abnormally high activity of RyR2. Growing evidence supports hyperphosphorylation of RyR2 at the CaMKII site S-2814 as a potential mechanism for altered RyR2 function. However, the specific molecular mechanisms underlying RyR2 hyperphosphorylation remain poorly understood.MicroRNAs are small noncoding RNAs that regulate protein expression by interfering with mRNAs of target genes. We recently reported that 2-fold overexpession of microRNA miR-1 enhances CaMKII-dependent RyR2 phosphorylation by disrupting protein phosphatase 2A scaffolding to the RyR2, resulting in increased activity of the channel and Ca-dependent afterdepolarizations in myocytes. In the present study, we used a canine model of nonischemic HF to test the hypothesis that the HF-related alterations in RyR2 phosphorylation levels are caused by a decrease in phosphatase activity localized to RyR2 due to enhanced expression of two most abundant muscle-specific microRNAs miR-1 and miR-133. qRT-PCR studies revealed that the levels of miR-1 and miR-133 were significantly increased in HF myocytes compared to controls (2 and 1.6 fold accordingly). Western blotting showed that PP2A regulatory (b56alpha) and catalytic subunits, specific targets of miR-1 and miR-133 validated by luciferase-reporter assay, were decreased in HF cells. Analysis using phospho-specific antibodies confirmed that RyR2 phosphorylation at Ser-2814 was significantly increased in HF myocytes compared to controls. CaMKII inhibitory peptide reduced the frequency of spontaneous Ca waves in paced current-clamped HF myocytes to low control values. These finding suggest that altered levels of major muscle-specific microRNAs contribute to abnormal RyR2 function in HF by depressing localized phosphatase activity to the channel, thus leading to excessive phosphorylation of RyR2s.