The potential of microRNAs (miRNA) as non-invasive diagnostic, prognostic, and predictive biomarkers, as well as therapeutic targets, has recently been recognized. Previous studies have highlighted the importance of consistency in the methodology used, but to our knowledge, no study has described the methodology of sample preparation and storage systematically with respect to miRNAs as blood biomarkers. The aim of this study was to investigate the stability of miRNAs in blood under various relevant clinical and research conditions: different collection tubes, storage at different temperatures, physical disturbance, as well as serial freeze-thaw cycles. Blood samples were collected from 12 healthy donors into different collection tubes containing anticoagulants, including EDTA, citrate and lithium-heparin, as well as into serum collection tubes. MiRNA stability was evaluated by measuring expression changes of miR-1, miR-21 and miR-29b at different conditions: varying processing time of whole blood (up to 72 hours (h)), long-term storage (9 months at -80°C), physical disturbance (1 and 8 h), as well as in a series of freeze/thaw cycles (1 and 4 times). Different collection tubes revealed comparable concentrations of miR-1, miR-21 and miR-29b. Tubes with lithium-heparin were found unsuitable for miRNA quantification. MiRNA levels were stable for at least 24 h at room temperature in whole blood, while separated fractions did show alterations within 24 h. There were significant changes in the miR-21 and miR-29b levels after 72 h incubation of whole blood at room temperature (p<0.01 for both). Both miR-1 and miR-21 showed decreased levels after physical disturbance for 8 h in separated plasma and miR-1 in serum whole blood, while after 1 h of disturbance no changes were observed. Storage of samples at -80°C extended the miRNA stability remarkably, however, miRNA levels in long-term stored (9 months) whole blood samples were significantly changed, which is in contrast to the plasma samples, where miR-21 or miR-29b levels were found to be stable. Repetitive (n = 4) freeze-thaw cycles resulted in a significant reduction of miRNA concentration both in plasma and serum samples. This study highlights the importance of proper and systematic sample collection and preparation when measuring circulating miRNAs, e.g., in context of clinical trials. We demonstrated that the type of collection tubes, preparation, handling and storage of samples should be standardized to avoid confounding variables influencing the results.

Background—: Congestive heart failure (CHF) causes atrial fibrotic remodeling, a substrate for atrial fibrillation (AF) maintenance. MicroRNA29 (miR29) targets extracellular matrix proteins. In the present study, we examined miR29b changes in patients with AF and/or CHF and in a CHF-related AF animal model and assessed its potential role in controlling atrial fibrous tissue production. Methods and Results—: Control dogs were compared with dogs subjected to ventricular tachypacing for 24 hours, 1 week, or 2 weeks to induce CHF. Atrial miR29b expression decreased within 24 hours in both whole atrial tissue and atrial fibroblasts (−87% and −92% versus control, respectively; p <0.001 for both) and remained decreased throughout the time course. Expression of miR29b extracellular matrix target genes collagen-1A1 (COL1A1), collagen-3A1 (COL3A1), and fibrillin increased significantly in CHF fibroblasts. Lentivirus-mediated miR29b knockdown in canine atrial fibroblasts (−68%; p <0.01) enhanced COL1A1, COL3A1, and fibrillin mRNA expression by 28% ( p <0.01), 19% ( p <0.05), and 20% ( p <0.05), respectively, versus empty virus–infected fibroblasts and increased COL1A1 protein expression by 90% ( p <0.05). In contrast, 3-fold overexpression of miR29b decreased COL1A1, COL3A1, and fibrillin mRNA by 65%, 62%, and 61% (all p <0.001), respectively, versus scrambled control and decreased COL1A1 protein by 60% ( p <0.05). MiR29b plasma levels were decreased in patients with CHF or AF (by 53% and 54%, respectively; both p <0.001) and were further decreased in patients with both AF and CHF (by 84%; p <0.001). MiR29b expression was also reduced in the atria of chronic AF patients (by 54% versus sinus rhythm; p <0.05). Adenoassociated viral–mediated knockdown of miR29b in mice significantly increased atrial COL1A1 mRNA expression and cardiac tissue collagen content. Conclusions—: MiR29 likely plays a role in atrial fibrotic remodeling and may have value as a biomarker and/or therapeutic target.

Background: Ibrutinib is a Bruton tyrosine kinase inhibitor with remarkable efficacy against B-cell cancers. Ibrutinib also increases the risk of atrial fibrillation (AF), which remains poorly understood. Methods: We performed electrophysiology studies on mice treated with ibrutinib to assess inducibility of AF. Chemoproteomic analysis of cardiac lysates identified candidate ibrutinib targets, which were further evaluated in genetic mouse models and additional pharmacological experiments. The pharmacovigilance database, VigiBase, was queried to determine whether drug inhibition of an identified candidate kinase was associated with increased reporting of AF. Results: We demonstrate that treatment of mice with ibrutinib for 4 weeks results in inducible AF, left atrial enlargement, myocardial fibrosis, and inflammation. This effect was reproduced in mice lacking Bruton tyrosine kinase, but not in mice treated with 4 weeks of acalabrutinib, a more specific Bruton tyrosine kinase inhibitor, demonstrating that AF is an off-target side effect. Chemoproteomic profiling identified a short list of candidate kinases that was narrowed by additional experimentation leaving CSK (C-terminal Src kinase) as the strongest candidate for ibrutinib-induced AF. Cardiac-specific Csk knockout in mice led to increased AF, left atrial enlargement, fibrosis, and inflammation, phenocopying ibrutinib treatment. Disproportionality analyses in VigiBase confirmed increased reporting of AF associated with kinase inhibitors blocking Csk versus non-Csk inhibitors, with a reporting odds ratio of 8.0 (95% CI, 7.3–8.7; P <0.0001). Conclusions: These data identify Csk inhibition as the mechanism through which ibrutinib leads to AF. Registration: URL: https://ww.clinicaltrials.gov ; Unique identifier: NCT03530215.