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Circulating cell-free microRNAs (miRNAs) represent a major reservoir for biomarker discovery. Unfortunately, their implementation in clinical practice is limited due to a profound lack of reproducibility. The great technical variability linked to major pre-analytical and analytical caveats makes the interpretation of circulating cell-free miRNA data challenging and leads to inconsistent findings. Additional efforts directed to standardization are fundamental. Several well-established protocols are currently used by independent groups worldwide. Nonetheless, there are some specific aspects in specimen collection and processing, sample handling, miRNA quantification, and data analysis that should be considered to ensure reproducibility of results. Here, we have addressed this challenge using an alternative approach. We have highlighted and discussed common pitfalls that negatively impact the robustness of circulating miRNA quantification and their application for clinical decision-making. Furthermore, we provide a checklist usable by investigators to facilitate and ensure the control of the whole miRNA quantification and analytical process. We expect that these recommendations improve the reproducibility of findings, and ultimately, facilitate the incorporation of circulating miRNA profiles into clinical practice as the next generation of disease biomarkers.

Despite promising findings, quantitative PCR (qPCR)-based tests for RNA quantification have experienced serious limitations in their clinical application. The noticeable lack of technical standardization remains a huge obstacle in the translation of qPCR-based tests. The incorporation of qPCR-based tests into the clinic will benefit from guidelines for clinical research assay validation. This will ultimately impact the clinical management of the patient, including diagnosis, prognosis, prediction, monitoring of the therapeutic response, and evaluation of toxicity. However, clear assay validation protocols for biomarker investigation in clinical trials using molecular assays are currently lacking. Here, we will focus on the necessary steps, including sample acquisition, processing and storage, RNA purification, target selection, assay design, and experimental design, that need to be taken toward the appropriate validation of qRT-PCR assays in clinical research. These recommendations can fill the gap between research use only (RUO) and in vitro diagnostics (IVD). Our contribution provides a tool for basic and clinical research for the development of validated assays in the intermediate steps of biomarker research. These guidelines are based on the current understanding and consensus within the EU-CardioRNA COST Action consortium (www. cardiorna.eu). Their applicability encompasses all clinical areas.

Prior studies show that glycogen synthase kinase 3 beta (GSK3 beta) contributes to cardiac ischemic injury and cardiac hypertrophy. GSK3 beta is constitutionally active and phosphorylation of GSK3 beta at serine 9 (S9) inactivates the kinase and promotes cellular growth. GSK3 beta is also phosphorylated at serine 389 (S389), but the significance of this phosphorylation in the heart is not known. We analyzed GSK3 beta S389 phosphorylation in diseased hearts and utilized overexpression of GSK3 beta carrying ser & RARR;ala mutations at S9 (S9A) and S389 (S389A) to study the biological function of constitutively active GSK3 beta in primary cardiomyocytes. We found that phosphorylation of GSK3 beta at S389 was increased in left ventricular samples from patients with dilated cardiomyopathy and ischemic cardiomyopathy, and in hearts of mice subjected to thoracic aortic constriction. Overexpression of either GSK3 beta S9A or S389A reduced the viability of cardiomyocytes subjected to hypoxia-reoxygenation. Overexpression of double GSK3 beta mutant (S9A/S389A) further reduced cardiomyocyte viability. Determination of protein synthesis showed that overexpression of GSK3 beta S389A or GSK3 beta S9A/S389A increased both basal and agonist-induced cardiomyocyte growth. Mechanistically, GSK3 beta S389A mutation was associated with activation of mTOR complex 1 signaling. In conclusion, our data suggest that phosphorylation of GSK3 beta at S389 enhances cardiomyocyte survival and protects from cardiomyocyte hypertrophy.

Heart failure causes significant morbidity and mortality worldwide. The understanding of heart failure pathomechanisms and options for treatment remain incomplete. Zebrafish has proven useful for modeling human heart diseases due to similarity of zebrafish and mammalian hearts, fast easily tractable development, and readily available genetic methods. Embryonic cardiac development is rapid and cardiac function is easy to observe and quantify. Reverse genetics, by using morpholinos and CRISPR-Cas9 to modulate gene function, make zebrafish a primary animal model for in vivo studies of candidate genes. Zebrafish are able to effectively regenerate their hearts following injury. However, less attention has been given to using zebrafish models to increase understanding of heart failure and cardiac remodeling, including cardiac hypertrophy and hyperplasia. Here we discuss using zebrafish to study heart failure and cardiac remodeling, and review zebrafish genetic, drug-induced and other heart failure models, discussing the advantages and weaknesses of using zebrafish to model human heart disease. Using zebrafish models will lead to insights on the pathomechanisms of heart failure, with the aim to ultimately provide novel therapies for the prevention and treatment of heart failure.

Prior studies show that glycogen synthase kinase 3β (GSK3β) contributes to cardiac ischemic injury and cardiac hypertrophy. GSK3β is constitutionally active and phosphorylation of GSK3β at serine 9 (S9) inactivates the kinase and promotes cellular growth. GSK3β is also phosphorylated at serine 389 (S389), but the significance of this phosphorylation in the heart is not known. We analyzed GSK3β S389 phosphorylation in diseased hearts and utilized overexpression of GSK3β carrying ser→ala mutations at S9 (S9A) and S389 (S389A) to study the biological function of constitutively active GSK3β in primary cardiomyocytes. We found that phosphorylation of GSK3β at S389 was increased in left ventricular samples from patients with dilated cardiomyopathy and ischemic cardiomyopathy, and in hearts of mice subjected to thoracic aortic constriction. Overexpression of either GSK3β S9A or S389A reduced the viability of cardiomyocytes subjected to hypoxia–reoxygenation. Overexpression of double GSK3β mutant (S9A/S389A) further reduced cardiomyocyte viability. Determination of protein synthesis showed that overexpression of GSK3β S389A or GSK3β S9A/S389A increased both basal and agonist-induced cardiomyocyte growth. Mechanistically, GSK3β S389A mutation was associated with activation of mTOR complex 1 signaling. In conclusion, our data suggest that phosphorylation of GSK3β at S389 enhances cardiomyocyte survival and protects from cardiomyocyte hypertrophy

Additional file 1. Detailed description of statistical analysis. E-Table 1: Comparison of latent class analysis models with different numbers of classes. E-Table 2: Pearson correlations of class defining variables with absolute correlations over 0.5. E-Table 3: List of variables included in multiple imputation and percentages of missing data. E-Table 4: All included variables for clustering. E-Table 5. Comparison of baseline characteristics between included and excluded patients. E-Table 6. Baseline of patients in class of 24-hour variable model. E-Table 7. Comparison of admission and 24-hour model classification. E-Figure 1: First two principal components of the variables used in the LCA, two class and three class model comparison. E-Figure 2: Heatmap of class assignments for the admission model across 31 imputations. E-Figure 3: Flowchart of patient inclusion. E-Figure 4: Standardized mean difference (SMD) plot of class defining variables of 24-hour model.

Aims The role of microRNAs has not been studied in cardiogenic shock. We examined the potential role of miR‐423‐5p level to predict mortality and associations of miR‐423‐5p with prognostic markers in cardiogenic shock. Methods and results We conducted a prospective multinational observational study enrolling consecutive cardiogenic shock patients. Blood samples were available for 179 patients at baseline to determine levels of miR‐423‐5p and other biomarkers. Patients were treated according to local practice. Main outcome was 90 day all‐cause mortality. Median miR‐423‐5p level was significantly higher in 90 day non‐survivors [median 0.008 arbitrary units (AU) (interquartile range 0.003–0.017) vs. 0.004 AU (0.002–0.009), P = 0.003]. miR‐423‐5p level above median was associated with higher lactate (median 3.7 vs. 2.4 mmol/L, P = 0.001) and alanine aminotransferase levels (median 68 vs. 35 IU/L, P

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WOS: 000474931400001, PubMed ID: 30934986, Cardiovascular disease (CVD) remains the leading cause of death worldwide and, despite continuous advances, better diagnostic and prognostic tools, as well as therapy, are needed. The human transcriptome, which is the set of all RNA produced in a cell, is much more complex than previously thought and the lack of dialogue between researchers and industrials and consensus on guidelines to generate data make it harder to compare and reproduce results. This European Cooperation in Science and Technology (COST) Action aims to accelerate the understanding of transcriptomics in CVD and further the translation of experimental data into usable applications to improve personalized medicine in this field by creating an interdisciplinary network. It aims to provide opportunities for collaboration between stakeholders from complementary backgrounds, allowing the functions of different RNAs and their interactions to be more rapidly deciphered in the cardiovascular context for translation into the clinic, thus fostering personalized medicine and meeting a current public health challenge. Thus, this Action will advance studies on cardiovascular transcriptomics, generate innovative projects, and consolidate the leadership of European research groups in the field. COST (European Cooperation in Science and Technology) is a funding organization for research and innovation networks (www.cost.eu)., COST (European Cooperation in Science and Technology) Action EU-CardioRNA [CA17129], This work is supported by COST (European Cooperation in Science and Technology) Action EU-CardioRNA CA17129. YD is the chair and CE is the vice-chair of the European COST Action CA17129, CardioRNA.

Ajuts: This work was supported by grants from the Finnish Foundation for Cardiovascular Research, Aarne Koskelo Foundation, Finnish Foundation for Laboratory Medicine, FinskaLäkaresällskapet, the Liv och Hälsa Foundation, and Finnish state funding for university-level research. The role of microRNAs has not been studied in cardiogenic shock. We examined the potential role of miR-423-5p level to predict mortality and associations of miR-423-5p with prognostic markers in cardiogenic shock. We conducted a prospective multinational observational study enrolling consecutive cardiogenic shock patients. Blood samples were available for 179 patients at baseline to determine levels of miR-423-5p and other biomarkers. Patients were treated according to local practice. Main outcome was 90 day all-cause mortality. Median miR-423-5p level was significantly higher in 90 day non-survivors [median 0.008 arbitrary units (AU) (interquartile range 0.003-0.017) vs. 0.004 AU (0.002-0.009), P = 0.003]. miR-423-5p level above median was associated with higher lactate (median 3.7 vs. 2.4 mmol/L, P = 0.001) and alanine aminotransferase levels (median 68 vs. 35 IU/L, P < 0.001) as well as lower cardiac index (1.8 vs. 2.4, P = 0.04) and estimated glomerular filtration rate (56 vs. 70 mL/min/1.73 m 2, P = 0.002). In Cox regression analysis, miR-423-5p level above median was associated with 90 day all-cause mortality independently of established risk factors of cardiogenic shock [adjusted hazard ratio 1.9 (95% confidence interval 1.2-3.2), P = 0.01]. In cardiogenic shock patients, above median level of miR-423-5p at baseline is associated with markers of hypoperfusion and seems to independently predict 90 day all-cause mortality.

Representative original blot of TGF-ß1 Western blotting. (PDF 163 kb)