Amniotic Fluid microRNA in Severe Twin-Twin Transfusion Syndrome Cardiomyopathy-Identification of Differences and Predicting Demise.

Twin-twin transfusion syndrome (TTTS) is a rare but serious cause of fetal cardiomyopathy with poorly understood pathophysiology and challenging prognostication. This study sought a nonbiased, comprehensive assessment of amniotic fluid (AF) microRNAs from TTTS pregnancies and associations of these miRNAs with clinical characteristics. For the discovery cohort, AF from ten fetuses with severe TTTS cardiomyopathy were selected and compared to ten normal singleton AF. Array panels assessing 384 microRNAs were performed on the discovery cohort and controls. Using a stringent q < 0.0025, arrays identified 32 miRNAs with differential expression. Top three microRNAs were miR-99b, miR-370 and miR-375. Forty distinct TTTS subjects were selected for a validation cohort. RT-PCR targeted six differentially-expressed microRNAs in the discovery and validation cohorts. Expression differences by array were confirmed by RT-PCR with high fidelity. The ability of these miRNAs to predict clinical differences, such as cardiac findings and later demise, was evaluated on TTTS subjects. Down-regulation of miRNA-127-3p, miRNA-375-3p and miRNA-886 were associated with demise. Our results indicate AF microRNAs have potential as a diagnostic and prognostic biomarker in TTTS. The top microRNAs have previously demonstrated roles in angiogenesis, cardiomyocyte stress response and hypertrophy. Further studies of the mechanism of actions and potential targets is warranted.

Serum circulating proteins from pediatric patients with dilated cardiomyopathy cause pathologic remodeling and cardiomyocyte stiffness.

Dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy and main indication for heart transplantation in children. Therapies specific to pediatric DCM remain limited due to lack of a disease model. Our previous study showed that treatment of neonatal rat ventricular myocytes (NRVMs) with serum from nonfailing or DCM pediatric patients activates the fetal gene program (FGP). Here we show that serum treatment with proteinase K prevents activation of the FGP, whereas RNase treatment exacerbates it, suggesting that circulating proteins, but not circulating miRNAs, promote these pathological changes. Evaluation of the protein secretome showed that midkine (MDK) is upregulated in DCM serum, and NRVM treatment with MDK activates the FGP. Changes in gene expression in serum-treated NRVMs, evaluated by next-generation RNA-Seq, indicated extracellular matrix remodeling and focal adhesion pathways were upregulated in pediatric DCM serum and in DCM serum-treated NRVMs, suggesting alterations in cellular stiffness. Cellular stiffness was evaluated by Atomic Force Microscopy, which showed an increase in stiffness in DCM serum-treated NRVMs. Of the proteins increased in DCM sera, secreted frizzled-related protein 1 (sFRP1) was a potential candidate for the increase in cellular stiffness, and sFRP1 treatment of NRVMs recapitulated the increase in cellular stiffness observed in response to DCM serum treatment. Our results show that serum circulating proteins promoted pathological changes in gene expression and cellular stiffness, and circulating miRNAs were protective against pathological changes.

Circulating cyclic adenosine monophosphate concentrations in milrinone treated paediatric patients after congenital heart surgery.

BACKGROUND: Milrinone is a phosphodiesterase type 3 inhibitor that results in a positive inotropic effect in the heart through an increase in cyclic adenosine monophosphate. The purpose of this study was to evaluate circulating cyclic adenosine monophosphate and milrinone concentrations in milrinone treated paediatric patients undergoing congenital heart surgery. METHODS: Single-centre prospective observational pilot study from January 2015 to December 2017 including children aged birth to 18 years. Milrinone and circulating cyclic adenosine monophosphate concentrations were measured at four time points through the first post-operative day and compared between patients with and without low cardiac output syndrome, defined using clinical and laboratory criteria. RESULTS: Fifty patients were included. Nine (18%) developed low cardiac output syndrome. For all patients, 22% had single ventricle heart disease. The density and distribution of cyclic adenosine monophosphate concentrations varied between those with and without low cardiac output syndrome but were not significantly different. Milrinone concentrations increased in all patients. Paired t-tests demonstrated an increase in circulating cyclic adenosine monophosphate concentrations during the post-operative period among patients without low cardiac output syndrome. CONCLUSIONS: In this prospective observational study, circulating cyclic adenosine monophosphate concentrations increased in those without low cardiac output syndrome during the first 24 post-operative hours and milrinone concentrations increased in all patients. Further study of the utility of cyclic adenosine monophosphate concentrations in milrinone treated patients is necessary.

Circulating microRNAs differentiate Kawasaki Disease from infectious febrile illnesses in childhood.

BACKGROUND: Kawasaki Disease (KD) is an acute vasculitis of unknown etiology in children that can lead to coronary artery lesions (CAL) in 25% of untreated patients. There is currently no diagnostic test for KD, and the clinical presentation is often difficult to differentiate from other febrile childhood illnesses. Circulating microRNAs (miRNAs) are small noncoding RNA molecules that control gene expression by inducing transcript degradation or by blocking translation. We hypothesize that the expression of circulating miRNAs will differentiate KD from non-KD febrile illnesses in children. METHODS: Circulating miRNA profiles from 84 KD patients and 29 non-KD febrile controls (7 viral and 22 bacterial infections) were evaluated. 3 ul of serum from each subject was submitted to 3 freeze/heat cycles to ensure miRNA release from microvesicles or interaction with serum proteins. miRNAs were reverse transcribed using a pool of primers specific for each miRNA. Real-time PCR reactions were performed in a 384 well plate containing sequence-specific primers and TaqMan probes in the ABI7900. '. RESULTS: KD patients (3.6 ± 2.2 yrs., 58% male) were found to have a unique circulating miRNA profile, including upregulation of miRNA-210-3p, -184, and -19a-3p (p < .0001), compared to non-KD febrile controls (8.5 ± 6.1 yrs., 72% male). CONCLUSIONS: Circulating miRNAs can differentiate KD from infectious febrile childhood diseases, supporting their potential as a diagnostic biomarker for KD.

Improved Detection of Circulating miRNAs in Serum and Plasma Following Rapid Heat/Freeze Cycling.

BACKGROUND: The measurement of circulating miRNAs has proven to be a powerful biomarker tool for several disease processes. Current protocols for the detection of miRNAs usually involve an RNA extraction step, requiring a substantial volume of patient serum or plasma to obtain sufficient input material. OBJECTIVE: Here, we describe a novel methodology that allows detection of a large number of miRNAs from a small volume of serum or plasma without the need for RNA extraction. METHODS: Three µl of serum or plasma was subjected to three cycles of high and low temperatures (heat/freeze cycles) followed by miRNA arrays. RESULTS: Our results indicate that miRNA detection following this process is highly reproducible when comparing multiple samples from the same subject. Moreover, this protocol increases the reproducibility of miRNA detection in samples that were previously subjected to multiple freeze-thaw cycles. Importantly, the detection of miRNAs from serum vs. plasma following heat/freeze cycling are highly comparable, indicating that this heat/freeze process effectively eliminates differences in detection between serum and plasma samples that have been reported using other sample preparation methodologies. CONCLUSION: We propose that this method is a potent alternative to current RNA extraction protocols, substantially reducing the amount of sample necessary for miRNA detection while simultaneously improving miRNA detection and reproducibility.

Sex-related differences in age-associated downregulation of human ventricular myocardial ß1-adrenergic receptors.

BACKGROUND: With increasing age, human ventricular myocardium exhibits selective downregulation of ß1-adrenergic receptors (ß1-ARs). We tested the hypothesis that sex differences exist in age-related changes in ß1-ARs. METHODS: Left (LV) and right (RV) ventricular tissue was obtained from 61 unplaceable potential organ donor hearts ages 1 to 71 years with no known cardiac history and from LVs removed from 56 transplant recipients with idiopathic dilated cardiomyopathy. ß1-AR and ß2-AR densities, the frequency of ß1-AR389 gene variants, and ß-AR function were determined. RESULTS: Sex had a marked effect on the age-related decrease in ß1-ARs. Female LVs had more pronounced downregulation (by 42% [p < 0.001] vs 22% [p = 0.21] in 31 male LVs) comparing the youngest (average age, 15.3 ± 5.5 years) to the oldest (average age, 50.8 ± 9.1 years) sub-groups. On regression analyses, female LVs exhibited a closer relationship between ß1-AR density and age (r = -0.78, p <0.001 vs r = -0.46, p = 0.009 in males), with a second-degree polynomial yielding the best fit. There was no statistically significant relationship of ß1-ARs to age in female or male idiopathic dilated cardiomyopathy LVs. CONCLUSIONS: Sex affects age-related ß-AR downregulation in normal human ventricles, with females exhibiting more profound decreases with increasing age. The curvilinear relationship between age and receptor density that plateaus around age 40 in women suggests an effect of sex hormones on ß1-AR expression in the human heart.