Quaking regulates circular RNA production in cardiomyocytes.

Circular RNAs (circRNAs) are a class of non-coding RNA molecules that are gaining increasing attention for their roles in various pathophysiological processes. The RNA-binding protein quaking (QKI) has been identified as a regulator of circRNA formation. In this study, we investigate the role of QKI in the formation of circRNAs in the heart by performing RNA-sequencing on Qki-knockout mice. Loss of QKI resulted in the differential expression of 17% of the circRNAs in adult mouse hearts. Interestingly, the majority of the QKI-regulated circRNAs (58%) were derived from genes undergoing QKI-dependent splicing, indicating a relationship between back-splicing and linear splicing. We compared these QKI-dependent circRNAs with those regulated by RBM20, another cardiac splicing factor essential for circRNA formation. We found that QKI and RBM20 regulate the formation of a distinct, but partially overlapping set of circRNAs in the heart. Strikingly, many shared circRNAs were derived from the Ttn gene, and they were regulated in an opposite manner. Our findings indicate that QKI not only regulates alternative splicing in the heart but also the formation of circRNAs.

Inhibition of minor intron splicing reduces Na+ and Ca2+ channel expression and function in cardiomyocytes.

Eukaryotic genomes contain a tiny subset of 'minor class' introns with unique sequence elements that require their own splicing machinery. These minor introns are present in certain gene families with specific functions, such as voltage-gated Na+ and voltage-gated Ca2+ channels. Removal of minor introns by the minor spliceosome has been proposed as a post-transcriptional regulatory layer, which remains unexplored in the heart. Here, we investigate whether the minor spliceosome regulates electrophysiological properties of cardiomyocytes by knocking down the essential minor spliceosome small nuclear snRNA component U6atac in neonatal rat ventricular myocytes. Loss of U6atac led to robust minor intron retention within Scn5a and Cacna1c, resulting in reduced protein levels of Nav1.5 and Cav1.2 channels. Functional consequences were studied through patch-clamp analysis, and revealed reduced Na+ and L-type Ca2+ currents after loss of U6atac. In conclusion, minor intron splicing modulates voltage-dependent ion channel expression and function in cardiomyocytes. This may be of particular relevance in situations in which minor splicing activity changes, such as in genetic diseases affecting minor spliceosome components, or in acquired diseases in which minor spliceosome components are dysregulated, such as heart failure.

Splicing factors in the heart: Uncovering shared and unique targets.

Alternative splicing generates specialized protein isoforms that allow the heart to adapt during development and disease. The recent discovery that mutations in the splicing factor RNA-binding protein 20 (RBM20) cause a severe form of familial dilated cardiomyopathy has sparked a great interest in alternative splicing in the field of cardiology. Since then, identification of splicing factors controlling alternative splicing in the heart has grown at a rapid pace. Despite the intriguing observation that a certain overlap exists between the targets of some splicing factors, an integrated and systematic analysis of their splicing networks is missing. Here, we compared the splicing networks of individual splicing factors by re-analyzing original RNA-sequencing data from eight previously published mouse models, in which a single splicing factor has been genetically deleted (i.e. HNRNPU, MBNL1/2, QKI, RBM20, RBM24, RBPMS, SRSF3, SRSF4). We show that key splicing events in Camk2d, Ryr2, Tpm1, Tpm2 and Pdlim5 require the combined action of the majority of these splicing factors. Additionally, we identified common targets and pathways among splicing factors, with the largest overlap between the splicing networks of MBNL, QKI and RBM24. We also re-analyzed a large-scale RNA-sequencing study on hearts of 128 heart failure patients. Here, we observed that MBNL1, QKI and RBM24 expression varied greatly. This variation in expression correlated with differential splicing of their downstream targets as found in mice, suggesting that aberrant splicing by MBNL1, QKI and RBM24 might contribute to the disease mechanism in heart failure.

Cardiac sequelae in athletes following COVID-19 vaccination: evidence and misinformation.

The recognition of myocarditis as a rare side effect of SARS-CoV-2 mRNA vaccination has sparked a global debate on vaccine safety, especially in the realm of sports. The main proposed mechanisms in the pathogenesis of COVID-19 mRNA vaccination-associated myocarditis (C-VAM) are based on the activation of the innate- and adaptive immune system against a susceptible immune-genetic background, including the recognition of mRNA as an antigen by the immune system, molecular mimicry between SARS-CoV-2 spike glycoprotein and cardiac tissue antigens and inflammatory sex-hormone signalling. The relatively younger age of the athlete population hypothetically constellates an increased risk of C-VAM. A subgroup analysis in individuals under 40 years revealed a low incidence of myocarditis following COVID-19 mRNA vaccination when compared to positive SARS-CoV-2 tests. No confirmed cases of athletes experiencing cardiac complications after mRNA vaccination have been reported. Most athletes only reported mild side effects after COVID-19 vaccination. A small but statistically significant decrease in maximal oxygen consumption in recreational athletes occurred after BNT162b2 mRNA booster vaccine administration. The clinical relevance and temporality of which remain to be determined. Many speculative social media reports attribute sudden cardiac arrest/death (SCA/D) in athletes to mRNA vaccination. Large media outlets have thoroughly debunked these claims. There is currently no evidence to support the claim that COVID-19 mRNA vaccination increases the risk of myocardial sequelae or SCA/D in athletes. However, specific vaccine regimen selection and timing may be appropriate to prevent detrimental performance effects.

Titin Circular RNAs Create a Back-Splice Motif Essential for SRSF10 Splicing.

BACKGROUND: TTN (Titin), the largest protein in humans, forms the molecular spring that spans half of the sarcomere to provide passive elasticity to the cardiomyocyte. Mutations that disrupt the TTN transcript are the most frequent cause of hereditary heart failure. We showed before that TTN produces a class of circular RNAs (circRNAs) that depend on RBM20 to be formed. In this study, we show that the back-splice junction formed by this class of circRNAs creates a unique motif that binds SRSF10 to enable it to regulate splicing. Furthermore, we show that one of these circRNAs (cTTN1) distorts both localization of and splicing by RBM20. METHODS: We calculated genetic constraint of the identified motif in 125 748 exomes collected from the gnomAD database. Furthermore, we focused on the highest expressed RBM20-dependent circRNA in the human heart, which we named cTTN1. We used shRNAs directed to the back-splice junction to induce selective loss of cTTN1 in human induced pluripotent stem cell-derived cardiomyocytes. RESULTS: Human genetics suggests reduced genetic tolerance of the generated motif, indicating that mutations in this motif might lead to disease. RNA immunoprecipitation confirmed binding of circRNAs with this motif to SRSF10. Selective loss of cTTN1 in human induced pluripotent stem cell-derived cardiomyocytes induced structural abnormalities, apoptosis, and reduced contractile force in engineered heart tissue. In line with its SRSF10 binding, loss of cTTN1 caused abnormal splicing of important cardiomyocyte SRSF10 targets such as MEF2A and CASQ2. Strikingly, loss of cTTN1 also caused abnormal splicing of TTN itself. Mechanistically, we show that loss of cTTN1 distorts both localization of and splicing by RBM20. CONCLUSIONS: We demonstrate that circRNAs formed from the TTN transcript are essential for normal splicing of key muscle genes by enabling splice regulators RBM20 and SRSF10. This shows that the TTN transcript also has regulatory roles, besides its well-known signaling and structural function. In addition, we demonstrate that the specific sequence created by the back-splice junction of these circRNAs has important functions. This highlights the existence of functionally important sequences that cannot be recognized as such in the human genome but provides an as-yet unrecognized source for functional sequence variation.

A deep learning approach identifies new ECG features in congenital long QT syndrome.

BACKGROUND: Congenital long QT syndrome (LQTS) is a rare heart disease caused by various underlying mutations. Most general cardiologists do not routinely see patients with congenital LQTS and may not always recognize the accompanying ECG features. In addition, a proportion of disease carriers do not display obvious abnormalities on their ECG. Combined, this can cause underdiagnosing of this potentially life-threatening disease. METHODS: This study presents 1D convolutional neural network models trained to identify genotype positive LQTS patients from electrocardiogram as input. The deep learning (DL) models were trained with a large 10-s 12-lead ECGs dataset provided by Amsterdam UMC and externally validated with a dataset provided by University Hospital Leuven. The Amsterdam dataset included ECGs from 10000 controls, 172 LQTS1, 214 LQTS2, and 72 LQTS3 patients. The Leuven dataset included ECGs from 2200 controls, 32 LQTS1, and 80 LQTS2 patients. The performance of the DL models was compared with conventional QTc measurement and with that of an international expert in congenital LQTS (A.A.M.W). Lastly, an explainable artificial intelligence (AI) technique was used to better understand the prediction models. RESULTS: Overall, the best performing DL models, across 5-fold cross-validation, achieved on average a sensitivity of 84 ± 2%, 90 ± 2% and 87 ± 6%, specificity of 96 ± 2%, 95 ± 1%, and 92 ± 4%, and AUC of 0.90 ± 0.01, 0.92 ± 0.02, and 0.89 ± 0.03, for LQTS 1, 2, and 3 respectively. The DL models were also shown to perform better than conventional QTc measurements in detecting LQTS patients. Furthermore, the performances held up when the DL models were validated on a novel external cohort and outperformed the expert cardiologist in terms of specificity, while in terms of sensitivity, the DL models and the expert cardiologist in LQTS performed the same. Finally, the explainable AI technique identified the onset of the QRS complex as the most informative region to classify LQTS from non-LQTS patients, a feature previously not associated with this disease. CONCLUSIONS: This study suggests that DL models can potentially be used to aid cardiologists in diagnosing LQTS. Furthermore, explainable DL models can be used to possibly identify new features for LQTS on the ECG, thus increasing our understanding of this syndrome.

Beneficial Effects of Cardiomyopathy-Associated Genetic Variants on Physical Performance: A Hypothesis-Generating Scoping Review.

BACKGROUND: Genetic variants associated with cardiomyopathies (CMPs) are prevalent in the general population. In young athletes, CMPs account for roughly a quarter of sudden cardiac death, with further unexplained clustering in specific sports. Consequently, most CMPs form a contraindication for competitive sports. We hypothesized that genetic variants might (paradoxically) improve physical performance early in life while impairing cardiac function later in life. METHODS: Systematic PubMed search was done to investigate whether genetic variants in genes associated with CMPs could be related to beneficial performance phenotypes. SUMMARY: In a limited number of studies (n = 6), 2,860 individuals/subjects with genetic variants were able to outperform those without said variants, as measured by running speed (∼38 m/min in heterozygous [HET] mice, n = 6, vs. ∼32 m/min in wild type [WT] mice, n = 7, p = 0.004) and distance (966 ± 169 km HET mice vs. 561 ± 144 km WT mice, p = 0.0035, n = 10), elite athlete status in endurance athletes (n = 1,672, p = 1.43 × 10-8), maximal oxygen uptake in elite athletes (absolute difference not provided, n = 32, p = 0.005), maximal oxygen uptake in unrelated individuals (n = 473, p = 0.0025), personal records in highly trained marathon runners (2:26:28 ± 0:06:23 min HET, n = 32, vs. 2:28:53 ± 0:05:50 min without polymorphism, n = 108, p = 0.020), and peripheral muscle force contraction in patients following a cardiac rehabilitation program (absolute values not provided, n = 260). Key Message: Beneficial effects in genetic variants associated with CMPs could hypothetically play a role in the selection of young athletes, consequently explaining the prevalence of such genetic variants in athletes and the general population.

shRNAs Targeting a Common KCNQ1 Variant Could Alleviate Long-QT1 Disease Severity by Inhibiting a Mutant Allele.

Long-QT syndrome type 1 (LQT1) is caused by mutations in KCNQ1. Patients heterozygous for such a mutation co-assemble both mutant and wild-type KCNQ1-encoded subunits into tetrameric Kv7.1 potassium channels. Here, we investigated whether allele-specific inhibition of mutant KCNQ1 by targeting a common variant can shift the balance towards increased incorporation of the wild-type allele to alleviate the disease in human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs). We identified the single nucleotide polymorphisms (SNP) rs1057128 (G/A) in KCNQ1, with a heterozygosity of 27% in the European population. Next, we determined allele-specificity of short-hairpin RNAs (shRNAs) targeting either allele of this SNP in hiPSC-CMs that carry an LQT1 mutation. Our shRNAs downregulated 60% of the A allele and 40% of the G allele without affecting the non-targeted allele. Suppression of the mutant KCNQ1 allele by 60% decreased the occurrence of arrhythmic events in hiPSC-CMs measured by a voltage-sensitive reporter, while suppression of the wild-type allele increased the occurrence of arrhythmic events. Furthermore, computer simulations based on another LQT1 mutation revealed that 60% suppression of the mutant KCNQ1 allele shortens the prolonged action potential in an adult cardiomyocyte model. We conclude that allele-specific inhibition of a mutant KCNQ1 allele by targeting a common variant may alleviate the disease. This novel approach avoids the need to design shRNAs to target every single mutation and opens up the exciting possibility of treating multiple LQT1-causing mutations with only two shRNAs.

Echocardiography protocol for early detection of cardiac dysfunction in childhood cancer survivors in the multicenter DCCSS LATER 2 CARD study: Design, feasibility, and reproducibility.

BACKGROUND: Cardiotoxicity is a well-known side effect after anthracyclines and chest radiotherapy in childhood cancer survivors (CCS). The DCCSS LATER 2 CARD (cardiology) study includes evaluation of echocardiographic measurements for early identification of CCS at highest risk of developing heart failure. This paper describes the design, feasibility, and reproducibility of the echocardiography protocol. METHODS: Echocardiograms from CCS and sibling controls were prospectively obtained at the participating centers and centrally analyzed. We describe the image acquisition, measurement protocol, and software-specific considerations for myocardial strain analyses. We report the feasibility of the primary outcomes of systolic and diastolic function, as well as reproducibility analyses in 30 subjects. RESULTS: We obtained 1,679 echocardiograms. Biplane ejection fraction (LVEF) measurement was feasible in 91% and 96% of CCS and siblings, respectively, global longitudinal strain (GLS) in 80% and 91%, global circumferential strain (GCS) in 86% and 89%, and ≥2 diastolic function parameters in 99% and 100%, right ventricle free wall strain (RVFWS) in 57% and 65%, and left atrial reservoir strain (LASr) in 72% and 79%. Intra-class correlation coefficients for inter-observer variability were 0.85 for LVEF, 0.76 for GLS, 0.70 for GCS, 0.89 for RVFWS and 0.89 for LASr. Intra-class correlation coefficients for intra-observer variability were 0.87 for LVEF, 0.82 for GLS, 0.82 for GCS, 0.85 for RVFWS and 0.79 for LASr. CONCLUSION: The DCCSS LATER 2 CARD study includes a protocolized echocardiogram, with feasible and reproducible primary outcome measurements. This ensures high-quality outcome data for prevalence estimates and for reliable comparison of cardiac function parameters.

circRNAprofiler: an R-based computational framework for the downstream analysis of circular RNAs.

BACKGROUND: Circular RNAs (circRNAs) are a newly appreciated class of non-coding RNA molecules. Numerous tools have been developed for the detection of circRNAs, however computational tools to perform downstream functional analysis of circRNAs are scarce. RESULTS: We present circRNAprofiler, an R-based computational framework that runs after circRNAs have been identified. It allows to combine circRNAs detected by multiple publicly available annotation-based circRNA detection tools and to analyze their expression, genomic context, evolutionary conservation, biogenesis and putative functions. CONCLUSIONS: Overall, the circRNA analysis workflow implemented by circRNAprofiler is highly automated and customizable, and the results of the analyses can be used as starting point for further investigation in the role of specific circRNAs in any physiological or pathological condition.

Rationale and design of the PRAETORIAN-COVID trial: A double-blind, placebo-controlled randomized clinical trial with valsartan for PRevention of Acute rEspiraTORy dIstress syndrome in hospitAlized patieNts with SARS-COV-2 Infection Disease.

There is much debate on the use of angiotensin receptor blockers (ARBs) in severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2)-infected patients. Although it has been suggested that ARBs might lead to a higher susceptibility and severity of SARS-CoV-2 infection, experimental data suggest that ARBs may reduce acute lung injury via blocking angiotensin-II-mediated pulmonary permeability, inflammation, and fibrosis. However, despite these hypotheses, specific studies on ARBs in SARS-CoV-2 patients are lacking. METHODS: The PRAETORIAN-COVID trial is a multicenter, double-blind, placebo-controlled 1:1 randomized clinical trial in adult hospitalized SARS-CoV-2-infected patients (n = 651). The primary aim is to investigate the effect of the ARB valsartan compared to placebo on the composite end point of admission to an intensive care unit, mechanical ventilation, or death within 14 days of randomization. The active-treatment arm will receive valsartan in a dosage titrated to blood pressure up to a maximum of 160 mg bid, and the placebo arm will receive matching placebo. Treatment duration will be 14 days, or until the occurrence of the primary end point or until hospital discharge, if either of these occurs within 14 days. The trial is registered at clinicaltrials.gov (NCT04335786, 2020). SUMMARY: The PRAETORIAN-COVID trial is a double-blind, placebo-controlled 1:1 randomized trial to assess the effect of valsartan compared to placebo on the occurrence of ICU admission, mechanical ventilation, and death in hospitalized SARS-CoV-2-infected patients. The results of this study might impact the treatment of SARS-CoV-2 patients globally.

Diagnostic tools for early detection of cardiac dysfunction in childhood cancer survivors: Methodological aspects of the Dutch late effects after childhood cancer (LATER) cardiology study.

BACKGROUND: Cancer therapy-related cardiac dysfunction and heart failure are major problems in long-term childhood cancer survivors (CCS). We hypothesize that assessment of more sensitive echo- and electrocardiographic measurements, and/or biomarkers will allow for improved recognition of patients with cardiac dysfunction before heart failure develops, and may also identify patients at lower risk for heart failure. OBJECTIVE: To describe the methodology of the Dutch LATER cardiology study (LATER CARD). METHODS: The LATER CARD study is a cross-sectional study in long-term CCS treated with (potentially) cardiotoxic cancer therapies and sibling controls. We will evaluate 1) the prevalence and associated (treatment related) risk factors of subclinical cardiac dysfunction in CCS compared to sibling controls and 2) the diagnostic value of echocardiography including myocardial strain and diastolic function parameters, blood biomarkers for cardiomyocyte apoptosis, oxidative stress, cardiac remodeling and inflammation and ECG or combinations of them in the surveillance for cancer therapy-related cardiac dysfunction. From 2017 to 2020 we expect to include 1900 CCS and 500 siblings. CONCLUSIONS: The LATER CARD study will provide knowledge on different surveillance modalities for detection of cardiac dysfunction in long-term CCS at risk for heart failure. The results of the study will enable us to improve long-term follow-up surveillance guidelines for CCS at risk for heart failure.

Cardiac circRNAs arise mainly from constitutive exons rather than alternatively spliced exons.

Circular RNAs (circRNAs) are a relatively new class of RNA molecules, and knowledge about their biogenesis and function is still in its infancy. It was recently shown that alternative splicing underlies the formation of circular RNAs (circRNA) arising from the Titin (TTN) gene. Since the main mechanism by which circRNAs are formed is still unclear, we hypothesized that alternative splicing, and in particular exon skipping, is a major driver of circRNA production. We performed RNA sequencing on human and mouse hearts, mapped alternative splicing events, and overlaid these with expressed circRNAs at exon-level resolution. In addition, we performed RNA sequencing on hearts of Rbm20 KO mice to address how important Rbm20-mediated alternative splicing is in the production of cardiac circRNAs. In human and mouse hearts, we show that cardiac circRNAs are mostly (∼90%) produced from constitutive exons and less (∼10%) from alternatively spliced exons. In Rbm20 KO hearts, we identified 38 differentially expressed circRNAs of which 12 were produced from the Ttn gene. Even though Ttn appeared the most prominent target of Rbm20 for circularization, we also detected Rbm20-dependent circRNAs arising from other genes including Fan1, Stk39, Xdh, Bcl2l13, and Sorbs1 Interestingly, only Ttn circRNAs seemed to arise from Rbm20-mediated skipped exons. In conclusion, cardiac circRNAs are mostly derived from constitutive exons, suggesting that these circRNAs are generated at the expense of their linear counterpart and that circRNA production impacts the accumulation of the linear mRNA.

Risk Prediction in Transition: MAGGIC Score Performance at Discharge and Incremental Utility of Natriuretic Peptides.

BACKGROUND: Risk stratification for hospitalized patients with heart failure (HF) remains a critical need. The Meta-Analysis Global Group in Chronic Heart Failure (MAGGIC) score is a robust model derived from patients with ambulatory HF. Its validity at the time of discharge and the incremental value of natriuretic peptides (NPs) in this setting is unclear. METHODS: This was a single-center study examining a total of 4138 patients with HF from 2 groups; hospital discharge patients from administrative data (n = 2503, 60.5%) and a prospective registry of patients with ambulatory HF (n = 1635, 39.5%). The ambulatory registry patients underwent N-terminal pro-B-type NP (BNP) measurement at enrollment, and in the hospitalize discharge cohort clinical BNP levels were abstracted. The primary endpoint was all-cause mortality within 1 year. MAGGIC score performance was compared between cohorts utilizing Cox regression and calibration plots. The incremental value of NPs was assessed using calculated area under the curve and net reclassification improvement (NRI). RESULTS: The hospitalized and ambulatory cohorts differed with respect to primary outcome (777 and 100 deaths, respectively), sex (52.1% vs 41.7% female) and race (35% vs 49.5% African American). The MAGGIC score showed poor discrimination of mortality risk in the hospital discharge (C statistic: 0.668, hazard ratio [HR]: 1.1 per point, 95% confidence interval [CI]: 0.652, 0.684) but fair discrimination in the ambulatory cohorts (C statistic: 0.784, HR: 1.16 per point, 95% CI: 0.74, 0.83), respectively, a difference that was statistically significant (P = .001 for C statistic, 0.002 for HR). Calibration assessment indicated that the slope and intercept (of MAGGIC-predicted to observed mortality) did not statistically differ from ideal in either cohort and did not differ between the cohorts (all P > .1). NP levels did not significantly improve prediction in the hospitalized cohort (P = .127) but did in the ambulatory cohort (C statistic: 0.784 [95% CI: 0.74, 0.83] vs 0.82 [95% CI: 0.78, 0.85]; P = .018) with a favorable NRI of 0.354 (95% CI: 0.202-0.469; P = .002). CONCLUSION: The MAGGIC score showed poor discrimination when used in patients with HF at hospital discharge, which was inferior to its performance in patients with ambulatory HF. Discrimination within the hospital discharge group was not improved by including hospital NP levels.

Therapeutic Delivery of miR-148a Suppresses Ventricular Dilation in Heart Failure.

Heart failure is preceded by ventricular remodeling, changes in left ventricular mass, and myocardial volume after alterations in loading conditions. Concentric hypertrophy arises after pressure overload, involves wall thickening, and forms a substrate for diastolic dysfunction. Eccentric hypertrophy develops in volume overload conditions and leads wall thinning, chamber dilation, and reduced ejection fraction. The molecular events underlying these distinct forms of cardiac remodeling are poorly understood. Here, we demonstrate that miR-148a expression changes dynamically in distinct subtypes of heart failure: while it is elevated in concentric hypertrophy, it decreased in dilated cardiomyopathy. In line, antagomir-mediated silencing of miR-148a caused wall thinning, chamber dilation, increased left ventricle volume, and reduced ejection fraction. Additionally, adeno-associated viral delivery of miR-148a protected the mouse heart from pressure-overload-induced systolic dysfunction by preventing the transition of concentric hypertrophic remodeling toward dilation. Mechanistically, miR-148a targets the cytokine co-receptor glycoprotein 130 (gp130) and connects cardiomyocyte responsiveness to extracellular cytokines by modulating the Stat3 signaling. These findings show the ability of miR-148a to prevent the transition of pressure-overload induced concentric hypertrophic remodeling toward eccentric hypertrophy and dilated cardiomyopathy and provide evidence for the existence of separate molecular programs inducing distinct forms of myocardial remodeling.

NT-proBNP (N-Terminal pro-B-Type Natriuretic Peptide)-Guided Therapy in Acute Decompensated Heart Failure: PRIMA II Randomized Controlled Trial (Can NT-ProBNP-Guided Therapy During Hospital Admission for Acute Decompensated Heart Failure Reduce Mortality and Readmissions?).

BACKGROUND: The concept of natriuretic peptide guidance has been extensively studied in patients with chronic heart failure (HF), with only limited success. The effect of NT-proBNP (N-terminal probrain natriuretic peptide)-guided therapy in patients with acute decompensated HF using a relative NT-proBNP target has not been investigated. This study aimed to assess whether NT-proBNP-guided therapy of patients with acute decompensated HF using a relative NT-proBNP target would lead to improved outcomes compared with conventional therapy. METHODS: We conducted a prospective randomized controlled trial to study the impact of in-hospital guidance for acute decompensated HF treatment by a predefined NT-proBNP target (>30% reduction from admission to discharge) versus conventional treatment. Patients with acute decompensated HF with NT-proBNP levels >1700 ng/L were eligible. After achieving clinical stability, 405 patients were randomized to either NT-proBNP-guided or conventional treatment (1:1). The primary end point was dual: a composite of all-cause mortality and HF readmissions in 180 days and the number of days alive out of the hospital in 180 days. Secondary end points were all-cause mortality within 180 days, HF readmissions within 180 days, and a composite of all-cause mortality and HF readmissions within 90 days. RESULTS: Significantly more patients in the NT-proBNP-guided therapy group were discharged with an NT-proBNP reduction of >30% (80% versus 64%, P=0.001). Nonetheless, NT-proBNP-guided therapy did not significantly improve the combined event rate for all-cause mortality and HF readmissions (hazard ratio, 0.96; 95% confidence interval, 0.72-1.37; P=0.99) or the median number of days alive outside of the hospital (178 versus 179 days for NT-proBNP versus conventional patients, P=0.39). Guided therapy also did not significantly improve any of the secondary end points. CONCLUSIONS: The PRIMA II trial (Can NT-ProBNP-Guided Therapy During Hospital Admission for Acute Decompensated Heart Failure Reduce Mortality and Readmissions?) demonstrates that the guidance of HF therapy to reach an NT-proBNP reduction of >30% after clinical stabilization did not improve 6-month outcomes. CLINICAL TRIAL REGISTRATION: URL: http://www.trialregister.nl. Unique identifier: NTR3279.

RBM20 Mutations Induce an Arrhythmogenic Dilated Cardiomyopathy Related to Disturbed Calcium Handling.

BACKGROUND: Mutations in RBM20 (RNA-binding motif protein 20) cause a clinically aggressive form of dilated cardiomyopathy, with an increased risk of malignant ventricular arrhythmias. RBM20 is a splicing factor that targets multiple pivotal cardiac genes, such as Titin (TTN) and CAMK2D (calcium/calmodulin-dependent kinase II delta). Aberrant TTN splicing is thought to be the main determinant of RBM20-induced dilated cardiomyopathy, but is not likely to explain the increased risk of arrhythmias. Here, we investigated the extent to which RBM20 mutation carriers have an increased risk of arrhythmias and explore the underlying molecular mechanism. METHODS: We compared clinical characteristics of RBM20 and TTN mutation carriers and used our previously generated Rbm20 knockout (KO) mice to investigate downstream effects of Rbm20-dependent splicing. Cellular electrophysiology and Ca2+ measurements were performed on isolated cardiomyocytes from Rbm20 KO mice to determine the intracellular consequences of reduced Rbm20 levels. RESULTS: Sustained ventricular arrhythmias were more frequent in human RBM20 mutation carriers than in TTN mutation carriers (44% versus 5%, respectively, P=0.006). Splicing events that affected Ca2+- and ion-handling genes were enriched in Rbm20 KO mice, most notably in the genes CamkIIδ and RyR2. Aberrant splicing of CamkIIδ in Rbm20 KO mice resulted in a remarkable shift of CamkIIδ toward the δ-A isoform that is known to activate the L-type Ca2+ current ( ICa,L). In line with this, we found an increased ICa,L, intracellular Ca2+ overload and increased sarcoplasmic reticulum Ca2+ content in Rbm20 KO myocytes. In addition, not only complete loss of Rbm20, but also heterozygous loss of Rbm20 increased spontaneous sarcoplasmic reticulum Ca2+ releases, which could be attenuated by treatment with the ICa,L antagonist verapamil. CONCLUSIONS: We show that loss of Rbm20 disturbs Ca2+ handling and leads to more proarrhythmic Ca2+ releases from the sarcoplasmic reticulum. Patients that carry a pathogenic RBM20 mutation have more ventricular arrhythmias despite a similar left ventricular function, in comparison with patients with a TTN mutation. Our experimental data suggest that RBM20 mutation carriers may benefit from treatment with an ICa,L blocker to reduce their arrhythmia burden.

Practical data handling pipeline improves performance of qPCR-based circulating miRNA measurements.

Since numerous miRNAs have been shown to be present in circulation, these so-called circulating miRNAs have emerged as potential biomarkers for disease. However, results of qPCR studies on circulating miRNA biomarkers vary greatly and many experiments cannot be reproduced. Missing data in qPCR experiments often occur due to off-target amplification, nonanalyzable qPCR curves and discordance between replicates. The low concentration of most miRNAs leads to most, but not all missing data. Therefore, failure to distinguish between missing data due to a low concentration and missing data due to randomly occurring technical errors partly explains the variation within and between otherwise similar studies. Based on qPCR kinetics, an analysis pipeline was developed to distinguish missing data due to technical errors from missing data due to a low concentration of the miRNA-equivalent cDNA in the PCR reaction. Furthermore, this pipeline incorporates a method to statistically decide whether concentrations from replicates are sufficiently concordant, which improves stability of results and avoids unnecessary data loss. By going through the pipeline's steps, the result of each measurement is categorized as "valid, invalid, or undetectable." Together with a set of imputation rules, the pipeline leads to more robust and reproducible data as was confirmed experimentally. Using two validation approaches, in two cohorts totaling 2214 heart failure patients, we showed that this pipeline increases both the accuracy and precision of qPCR measurements. In conclusion, this statistical data handling pipeline improves the performance of qPCR studies on low-expressed targets such as circulating miRNAs.