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Background: Ventricular repolarization time (ECG QT and JT intervals) is associated with malignant arrhythmia. Genome-wide association studies have identified 230 independent loci for QT and JT; however, 50% of their heritability remains unexplained. Previous work supports a causal effect of lower serum calcium concentrations on longer ventricular repolarization time. We hypothesized calcium interactions with QT and JT variant associations could explain a proportion of the missing heritability., Methods and Results: We performed genome-wide calcium interaction analyses for QT and JT intervals. Participants were stratified by their calcium level relative to the study distribution (top or bottom 20%). We performed a 2-stage analysis (genome-wide discovery [N=62 532] and replication [N=59 861] of lead variants) and a single-stage genome-wide meta-analysis (N=122 393, [European ancestry N=117 581, African ancestry N=4812]). We also calculated 2-degrees of freedom joint main and interaction and 1-degree of freedom interaction P values. In 2-stage and single-stage analyses, 50 and 98 independent loci, respectively, were associated with either QT or JT intervals (2-degrees of freedom joint main and interaction P value <5×10 -8 ). No lead variant had a significant interaction result after correcting for multiple testing and sensitivity analyses provided similar findings. Two loci in the single-stage meta-analysis were not reported previously ( SPPL2B and RFX6 )., Conclusions: We have found limited support for an interaction effect of serum calcium on QT and JT variant associations despite sample sizes with suitable power to detect relevant effects. Therefore, such effects are unlikely to explain a meaningful proportion of the heritability of QT and JT, and factors including rare variation and other environmental interactions need to be considered.

Polymorphisms in the cholesteryl ester transfer protein ( CETP ) gene are known to be strongly associated with increased cardiovascular risk, primarily through their effects on the lipid profile and consequently on atherosclerotic risk. The acute heart rate response (AHRR) to physical activity is closely related to individual cardiovascular health. This study aimed to investigate the effect of CETP gene polymorphisms on AHRR. Our analysis examines the association of five single nucleotide polymorphisms (SNPs; rs1532624, rs5882, rs708272, rs7499892, and rs9989419) and their haplotypes (H) in the CETP gene with AHRR in 607 people from the Hungarian population. Individual AHRR in the present study was assessed using the YMCA 3-min step test and was estimated as the difference between resting and post-exercise heart rate, i.e., delta heart rate (ΔHR). To exclude the direct confounding effect of the CETP gene on the lipid profile, adjustments for TG and HDL-C levels, next to conventional risk factors, were applied in the statistical analyses. Among the examined five SNPs, two showed a significant association with lower ΔHR (rs1532624-C dominant : B = -8.41, p < 0.001; rs708272-G dominant : B = -8.33, p < 0.001) and reduced the risk of adverse AHRR (rs1532624-C dominant : OR = 0.44, p = 0.004; rs708272-G dominant : OR = 0.43, p = 0.003). Among the ten haplotypes, two showed significant association with lower ΔHR (H3-CAGCA: B = -6.81, p = 0.003; H9-CGGCG: B = -14.64, p = 0.015) and lower risk of adverse AHRR (H3-CAGCA: OR = 0.58, p = 0.040; H9-CGGCG: OR = 0.05, p = 0.009) compared to the reference haplotype (H1-AGACG). Our study is the first to report a significant association between CETP gene polymorphisms and AHRR. It also confirms that the association of the CETP gene with cardiovascular risk is mediated by changes in heart rate in response to physical activity, in addition to its effect on lipid profile.

Background: CaM (calmodulin)-mediated long-QT syndrome is a genetic arrhythmia disorder (calmodulinopathies) characterized by a high prevalence of life-threatening ventricular arrhythmias occurring early in life. Three distinct genes ( CALM1 , CALM2 , and CALM3 ) encode for the identical CaM protein. Conventional pharmacotherapies fail to adequately protect against potentially lethal cardiac events in patients with calmodulinopathy., Methods: Five custom-designed CALM1 -, CALM2 -, and CALM3 -targeting short hairpin RNAs (shRNAs) were tested for knockdown (KD) efficiency using TSA201 cells and reverse transcription-quantitative polymerase chain reaction. A dual-component suppression and replacement (SupRep) CALM gene therapy (CALM-SupRep) was created by cloning into a single construct CALM1 -, CALM2 -, and CALM3-specific shRNAs that produce KD (suppression) of each respective gene and a shRNA-immune CALM1 cDNA (replacement). CALM1-F142L, CALM2-D130G, and CALM3-D130G induced pluripotent stem cell-derived CMs were generated from patients with CaM-mediated long-QT syndrome. A voltage-sensing dye was used to measure action potential duration at 90% repolarization (APD90)., Results: Following shRNA KD efficiency testing, a candidate shRNA was identified for CALM1 (86% KD), CALM2 (71% KD), and CALM3 (94% KD). The APD90 was significantly prolonged in CALM2-D130G (647±9 ms) compared with CALM2-WT (359±12 ms; P <0.0001). Transfection with CALM -SupRep shortened the average APD90 of CALM2-D130G to 457±19 ms (66% attenuation; P <0.0001). Additionally, transfection with CALM -SupRep shortened the APD90 of CALM1-F142L (665±9 to 410±15 ms; P <0.0001) and CALM3-D130G (978±81 to 446±6 ms; P <0.001)., Conclusions: We provide the first proof-of-principle suppression-replacement gene therapy for CaM-mediated long-QT syndrome. The CALM-SupRep gene therapy shortened the pathologically prolonged APD90 in CALM1 -, CALM2 -, and CALM3-variant CaM-mediated long-QT syndrome induced pluripotent stem cell-derived CM lines. The single CALM-SupRep construct may be able to treat all calmodulinopathies, regardless of which of the 3 CaM-encoding genes are affected., Competing Interests: Dr Ackerman is a consultant for Abbott, Boston Scientific, Bristol Myers Squibb, Daiichi Sankyo, Invitae, Medtronic, Tenaya Therapeutics, and UpToDate. Dr Ackerman and Mayo Clinic are involved in an equity/royalty relationship with AliveCor, Anumana, ARMGO Pharma, Pfizer, and Thryv Therapeutics. However, none of these entities have contributed to this study in any manner. The other authors report no conflicts.

Given that anxiety disorders (AD) are associated with reduced vagally-mediated heart rate variability (HRV), genetic variants related to HRV may provide insight into anxiety etiology. This study used polygenic risk scores (PRS) to explore the genetic overlap between AD and HRV, and investigated whether HRV-related polymorphisms influence anxiety risk. Resting vagally-mediated HRV was measured using a wearable device in 188 European individuals (AD=101, healthy controls=87). AD PRS was tested for association with resting HRV, and HRV PRS for association with AD. We also investigated 15 significant hits from an HRV genome-wide association study (GWAS) for association with resting HRV and AD and if this association is mediated through resting HRV. The AD PRS and HRV PRS showed nominally significant associations with resting HRV and anxiety disorders, respectively. HRV GWAS variants associated with resting HRV were rs12980262 (NDUFA11), rs2680344 (HCN4), rs4262 and rs180238 (GNG11), and rs10842383 (LINC00477). Mediation analyses revealed that NDUFA11 rs12980262 A-carriers and GNG11 rs180238 and rs4262 C-carriers had higher anxiety risk through lower HRV. This study supports an anxiety-HRV genetic relationship, with HRV-related genetic variants translating to AD. This study encourages exploration of HRV genetics to understand mechanisms and identify novel treatment targets for anxiety., Competing Interests: Declaration of competing interest JLK is a member of the Scientific Advisory Board of Myriad Neuroscience. JLK, AKT, and CCZ are authors on several patents relating to pharmacogenetic tests for psychiatric medications. The remaining authors have no conflicts of interest to declare., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Aims: Patients with mutations in SCN5A encoding NaV1.5 often display variable severity of electrical and structural alterations, but the underlying mechanisms are not fully elucidated. We here investigate the combined modulatory effect of genetic background and age on disease severity in the Scn5a1798insD/+ mouse model., Methods and Results: In vivo electrocardiogram and echocardiograms, ex vivo electrical and optical mapping, and histological analyses were performed in adult (2-7 months) and aged (8-28 months) wild-type (WT) and Scn5a1798insD/+ (mutant, MUT) mice from the FVB/N and 129P2 inbred strains. Atrio-ventricular (AV) conduction, ventricular conduction, and ventricular repolarization are modulated by strain, genotype, and age. An aging effect was present in MUT mice, with aged MUT mice of both strains showing prolonged QRS interval and right ventricular (RV) conduction slowing. 129P2-MUT mice were severely affected, with adult and aged 129P2-MUT mice displaying AV and ventricular conduction slowing, prolonged repolarization, and spontaneous arrhythmias. In addition, the 129P2 strain appeared particularly susceptible to age-dependent electrical, functional, and structural alterations including RV conduction slowing, reduced left ventricular (LV) ejection fraction, RV dilatation, and myocardial fibrosis as compared to FVB/N mice. Overall, aged 129P2-MUT mice displayed the most severe conduction defects, RV dilatation, and myocardial fibrosis, in addition to the highest frequency of spontaneous arrhythmia and inducible arrhythmias., Conclusion: Genetic background and age both modulate disease severity in Scn5a1798insD/+ mice and hence may explain, at least in part, the variable disease expressivity observed in patients with SCN5A mutations. Age- and genetic background-dependent development of cardiac structural alterations furthermore impacts arrhythmia risk. Our findings therefore emphasize the importance of continued assessment of cardiac structure and function in patients carrying SCN5A mutations., Competing Interests: Conflict of interest: none declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Aims: Short QT syndrome type 3 (SQTS3) is a rare arrhythmogenic disease caused by gain-of-function mutations in KCNJ2, the gene coding the inward rectifier potassium channel Kir2.1. We used a multidisciplinary approach and investigated arrhythmogenic mechanisms in an in-vivo model of de-novo mutation Kir2.1E299V identified in a patient presenting an extremely abbreviated QT interval and paroxysmal atrial fibrillation., Methods and Results: We used intravenous adeno-associated virus-mediated gene transfer to generate mouse models, and confirmed cardiac-specific expression of Kir2.1WT or Kir2.1E299V. On ECG, the Kir2.1E299V mouse recapitulated the QT interval shortening and the atrial-specific arrhythmia of the patient. The PR interval was also significantly shorter in Kir2.1E299V mice. Patch-clamping showed extremely abbreviated action potentials in both atrial and ventricular Kir2.1E299V cardiomyocytes due to a lack of inward-going rectification and increased IK1 at voltages positive to -80 mV. Relative to Kir2.1WT, atrial Kir2.1E299V cardiomyocytes had a significantly reduced slope conductance at voltages negative to -80 mV. After confirming a higher proportion of heterotetrameric Kir2.x channels containing Kir2.2 subunits in the atria, in-silico 3D simulations predicted an atrial-specific impairment of polyamine block and reduced pore diameter in the Kir2.1E299V-Kir2.2WT channel. In ventricular cardiomyocytes, the mutation increased excitability by shifting INa activation and inactivation in the hyperpolarizing direction, which protected the ventricle against arrhythmia. Moreover, Purkinje myocytes from Kir2.1E299V mice manifested substantially higher INa density than Kir2.1WT, explaining the abbreviation in the PR interval., Conclusion: The first in-vivo mouse model of cardiac-specific SQTS3 recapitulates the electrophysiological phenotype of a patient with the Kir2.1E299V mutation. Kir2.1E299V eliminates rectification in both cardiac chambers but protects against ventricular arrhythmias by increasing excitability in both Purkinje-fiber network and ventricles. Consequently, the predominant arrhythmias are supraventricular likely due to the lack of inward rectification and atrial-specific reduced pore diameter of the Kir2.1E299V-Kir2.2WT heterotetramer., Competing Interests: Conflict of interest: none declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Background: Observational studies have demonstrated that a higher resting heart rate (RHR) is associated with an increased risk of dementia. However, it is not clear whether the association is causal. This study aimed to determine the causal effects of higher genetically predicted RHR on the risk of dementia., Methods: We performed a two-sample Mendelian randomization analysis to investigate the causal effect of higher genetically predicted RHR on Alzheimer's disease (AD) using summary statistics from genome-wide association studies. The generalized summary Mendelian randomization (GSMR) analysis was used to analyze the corresponding effects of RHR on following different outcomes: 1) diagnosis of AD (International Genomics of Alzheimer's Project), 2) family history (maternal and paternal) of AD from UK Biobank, 3) combined meta-analysis including these three GWAS results. Further analyses were conducted to determine the possibility of reverse causal association by adjusting for RHR modifying medication., Results: The results of GSMR showed no significant causal effect of higher genetically predicted RHR on the risk of AD (β GSMR = 0.12, P = 0.30). GSMR applied to the maternal family history of AD (β GSMR = -0.18, P = 0.13) and to the paternal family history of AD (β GSMR = -0.14, P = 0.39) showed the same results. Furthermore, the results were robust after adjusting for RHR modifying drugs (β GSMR = -0.03, P = 0.72)., Conclusion: Our study did not find any evidence that supports a causal effect of RHR on dementia. Previous observational associations between RHR and dementia are likely attributed to the correlation between RHR and other cardiovascular diseases., Competing Interests: Kenta Nakai is an Academic Editor for PeerJ., (© 2024 Chen et al.)

The acute heart rate response (AHRR) to physical activity, which refers to the change in heart rate during and after exercise, has been associated with cardiovascular and all-cause mortality. Previous studies have shown that AHRR is significantly determined by genetics in addition to environmental and lifestyle factors. The aim of this study was to investigate the genetic background of AHRR by analysing ten single nucleotide polymorphisms (SNPs) associated with leisure-time physical activity (LTPA) in 620 samples from the Hungarian population. The AHRR can be characterised as the difference between post-exercise and resting heart rate, i.e., the delta heart rate (ΔHR) defined by the YMCA 3 min step test, with a lower value indicating better cardiovascular fitness. The association of SNPs with ΔHR was analysed both separately and in combination using an optimised polygenic score (oPGS). The results showed that five SNPs (rs10252228, rs459465, rs6022999, rs8097348, and rs12405556) had at least nominally significant ( p < 0.05) individual associations with ΔHR. After optimizing the PGS, a cumulative effect was observed for eight SNPs (rs6022999, rs12405556, rs459465, rs10252228, rs8097348, rs10887741, rs12612420, and rs7023003) that had a strong and statistically significant association with ΔHR (B = -2.51, 95% CI: -3.46--1.76; p = 2.99 × 10 -9 ). Of the four main domains of physical activity, the oPGS showed a significant positive association only with LTPA (B = 84.60; 95%CI: 25.23-143.98; p = 0.005). In conclusion, our results suggest that the SNPs we investigated influence individual leisure-time physical activity, mediated by their effects on the acute heart rate response.

Objective: Previous studies have shown that emotional disorders are negatively associated with heart rate variability (HRV), but the potential causal relationship between genetic susceptibility to emotional disorders and HRV remains unclear. We aimed to perform a Mendelian randomization (MR) study to investigate the potential association between emotional disorders and HRV., Methods: The data used for this study were obtained from publicly available genome-wide association study datasets. Five models, including the inverse variance weighted model (IVW), the weighted median estimation model (WME), the weighted model-based method (WM), the simple model (SM) and the MR-Egger regression model (MER), were utilized for MR. The leave-one-out sensitivity test, MR pleiotropy residual sum and outlier test (MR-PRESSO) and Cochran's Q test were used to confirm heterogeneity and pleiotropy., Results: MR analysis revealed that genetic susceptibility to broad depression was negatively correlated with HRV (pvRSA/HF) (OR = 0.380, 95% CI 0.146-0.992; p = 0.048). However, genetic susceptibility to irritability was positively correlated with HRV (pvRSA/HF, SDNN) (OR = 2.017, 95% CI 1.152-3.534, p = 0.008) (OR = 1.154, 95% CI 1.000-1.331, p = 0.044). Genetic susceptibility to anxiety was positively correlated with HRV (RMSSD) (OR = 2.106, 95% CI 1.032-4.299; p = 0.041). No significant directional pleiotropy or heterogeneity was detected. The accuracy and robustness of these findings were confirmed through a sensitivity analysis., Conclusions: Our MR study provides genetic support for the causal effects of broad depression, irritable mood, and anxiety on HRV., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Luo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

We used N -ethyl- N -nitrosurea-induced germline mutagenesis combined with automated meiotic mapping to identify specific systolic blood pressure (SBP) and heart rate (HR) determinant loci. We analyzed 43,627 third-generation (G3) mice from 841 pedigrees to assess the effects of 45,378 variant alleles within 15,760 genes, in both heterozygous and homozygous states. We comprehensively tested 23% of all protein-encoding autosomal genes and found 87 SBP and 144 HR (with 7 affecting both) candidates exhibiting detectable hypomorphic characteristics. Unexpectedly, only 18 of the 87 SBP genes were previously known, while 26 of the 144 genes linked to HR were previously identified. Furthermore, we confirmed the influence of two genes on SBP regulation and three genes on HR control through reverse genetics. This underscores the importance of our research in uncovering genes associated with these critical cardiovascular risk factors and illustrate the effectiveness of germline mutagenesis for defining key determinants of polygenic phenotypes that must be studied in an intact organism.

Background: The causal relationship between heart rate variability and cardiovascular diseases and the associated events is still unclear, and the conclusions of current studies are inconsistent. We aimed to explore the relationship between heart rate variability and cardiovascular diseases and the associated events with the Mendelian randomization study., Methods: We selected normal-to-normal inter-beat intervals (SDNN), root mean square of the successive differences of inter-beat intervals (RMSSD) and peak-valley respiratory sinus arrhythmia or high-frequency power (pvRSA/HF) as the three sets of instrumental variables for heart rate variability. The outcome for cardiovascular diseases included essential hypertension, heart failure, angina pectoris, myocardial infarction, nonischemic cardiomyopathy and arrhythmia. Cardiac arrest, cardiac death and major coronary heart disease event were defined as the related events of cardiovascular diseases. The data for exposures and outcomes were derived from publicly available genome-wide association studies. Inverse variance weighted was used for the main causal estimation. Analyses of heterogeneity and pleiotropy were conducted using the Cochran Q test of Inverse variance weighted and MR-Egger, leave-one-out analysis, and MR-Pleiotropy Residual Sum and Outlier methods., Results: The Inverse variance weighted method indicated that genetically predicted pvRSA/HF was associated with the increased risk of cardiac arrest (odds ratio 2.02, 95% confidence interval 1.25-3.28, p = .004). The results were free of heterogeneity and pleiotropy. There were no outliers and the leave-one-out analysis proved that the results were reliable., Conclusions: This study provides genetic evidence that pvRSA/HF is causally related to cardiac arrest., (© 2023 Stichting European Society for Clinical Investigation Journal Foundation. Published by John Wiley & Sons Ltd.)

Low heart rate variability (HRV) has been widely reported as a predictor for increased mortality. However, the molecular mechanisms are poorly understood. Therefore, this study aimed to identify novel genetic loci associated with HRV and assess the association of phenotypic HRV and genetically predicted HRV with mortality. In a GWAS of 46,075 European ancestry individuals from UK biobank, we identified 17 independent genome-wide significant genetic variants in 16 loci associated with HRV traits. Notably, eight of these loci (RNF220, GNB4, LINCR-002, KLHL3/HNRNPA0, CHRM2, KCNJ5, MED13L, and C160rf72) have not been reported previously. In a prospective phenotypic relationship between HRV and mortality during a median follow-up of seven years, individuals with lower HRV had higher risk of dying from any cause. Genetically predicted HRV, as determined by the genetic risk scores, was not associated with mortality. To the best of our knowledge, the findings provide novel biological insights into the mechanisms underlying HRV. These results also underline the role of the cardiac autonomic nervous system, as indexed by HRV, in predicting mortality., (© 2023. Springer Nature Limited.)

Resting heart rate is associated with cardiovascular diseases and mortality in observational and Mendelian randomization studies. The aims of this study are to extend the number of resting heart rate associated genetic variants and to obtain further insights in resting heart rate biology and its clinical consequences. A genome-wide meta-analysis of 100 studies in up to 835,465 individuals reveals 493 independent genetic variants in 352 loci, including 68 genetic variants outside previously identified resting heart rate associated loci. We prioritize 670 genes and in silico annotations point to their enrichment in cardiomyocytes and provide insights in their ECG signature. Two-sample Mendelian randomization analyses indicate that higher genetically predicted resting heart rate increases risk of dilated cardiomyopathy, but decreases risk of developing atrial fibrillation, ischemic stroke, and cardio-embolic stroke. We do not find evidence for a linear or non-linear genetic association between resting heart rate and all-cause mortality in contrast to our previous Mendelian randomization study. Systematic alteration of key differences between the current and previous Mendelian randomization study indicates that the most likely cause of the discrepancy between these studies arises from false positive findings in previous one-sample MR analyses caused by weak-instrument bias at lower P-value thresholds. The results extend our understanding of resting heart rate biology and give additional insights in its role in cardiovascular disease development., (© 2023. The Author(s).)

Observational studies frequently report phenotypic associations between low resting heart rate (RHR) and higher levels of antisocial behaviour (ASB), although it remains unclear whether this relationship reflects causality. To triangulate evidence, we conducted two-sample univariable Mendelian randomisation (MR), multivariable MR and linkage disequilibrium score regression (LDSC) analyses. Genetic data were accessed from published genome-wide association studies (GWAS) for RHR (n = 458,835) and ASB (n = 85,359) for the univariable analyses, along with a third GWAS for heart rate variability (HRV; n = 53,174) for all other analyses. Genome-wide significant (p < 5 × 10 -8 ) single-nucleotide polymorphisms associated with RHR (n = 278) were selected as instrumental variables and the outcome was a composite measure of ASB. No causal association was observed between RHR and ASB (B IVW  =  - 0.0004, p = 0.841). The multivariable MR analyses including RHR and HRV also suggested no causal associations (B IVW  = 0.016, p = 0.914) and no genetic correlations between the heart rate measures and ASB were observed using LDSC (r g  = 0.057, p = 0.169). Sensitivity analyses suggested that our results are not likely to be affected by heterogeneity, pleiotropic effects, or reverse causation. These findings suggest that individual differences in autonomic nervous system functioning indexed by RHR are not likely to directly contribute to the development of ASB. Therefore, previously observed associations between RHR and ASB may arise from confounding, reverse causation, and/or additional study characteristics. Further causally informative longitudinal research is required to confirm our findings, and caution should be applied when using measures of RHR in interventions targeting ASB., (© 2023. The Author(s).)

Aims: To determine whether the known single nucleotide polymorphisms in adrenoreceptor associated genes affect the haemodynamic response to dobutamine in critically ill neonates., Methods: Alleles in the known genetic single nucleotide polymorphisms in β1- and β2-adrenoceptor (AR) genes and Gs protein α-subunit gene (GNAS) possibly affecting inotropic effect were identified in patients of neonatal dobutamine pharmacokinetic-pharmacodynamic study. Linear mixed-effect models were used to describe the effect of genetic polymorphisms to heart rate (HR), left ventricular output (LVO) and right ventricular output (RVO) during dobutamine treatment., Results: Twenty-six neonates (5 term, 21 preterm) were studied. Dobutamine plasma concentration and exposure time respective HR (adjusted to gestational age) is dependent on β1-AR Arg389Gly polymorphism so that in G/G (Gly) homozygotes and G/C heterozygotes dobutamine increases HR more than in C/C (Arg) homozygotes, with parameter estimate (95% CI) of 38.3 (15.8-60.7) beats/min per AUC of 100 μg L -1  h, P = .0008. LVO (adjusted to antenatal glucocorticoid administration and illness severity) and RVO (adjusted to gestational age and illness severity) is dependent on GNAS c.393C > T polymorphism so that in T/T homozygotes and C/T heterozygotes but not in C/C homozygotes LVO and RVO increase with dobutamine treatment, 24.5 (6.2-42.9) mL kg -1  min -1 per AUC of 100 μg L -1  h, P = .0095 and 33.2 (12.1-54.3) mL kg -1  min -1 per AUC of 100 μg L -1  h, P = .0025, respectively., Conclusion: In critically ill neonates, β1-AR Arg389Gly and GNAS c.393C > T polymorphisms may play a role in the haemodynamic response to dobutamine during the first hours and days of life., (© 2022 British Pharmacological Society.)

Background: Both elevated and low resting heart rates are associated with atrial fibrillation (AF), suggesting a U-shaped relationship. However, evidence for a U-shaped causal association between genetically-determined resting heart rate and incident AF is limited. We investigated potential directional changes of the causal association between genetically-determined resting heart rate and incident AF., Method and Results: Seven cohorts of the AFGen consortium contributed data to this meta-analysis. All participants were of European ancestry with known AF status, genotype information, and a heart rate measurement from a baseline electrocardiogram (ECG). Three strata of instrumental variable-free resting heart rate were used to assess possible non-linear associations between genetically-determined resting heart rate and the logarithm of the incident AF hazard rate: <65; 65-75; and >75 beats per minute (bpm). Mendelian randomization analyses using a weighted resting heart rate polygenic risk score were performed for each stratum. We studied 38,981 individuals (mean age 59±10 years, 54% women) with a mean resting heart rate of 67±11 bpm. During a mean follow-up of 13±5 years, 4,779 (12%) individuals developed AF. A U-shaped association between the resting heart rate and the incident AF-hazard ratio was observed. Genetically-determined resting heart rate was inversely associated with incident AF for instrumental variable-free resting heart rates below 65 bpm (hazard ratio for genetically-determined resting heart rate, 0.96; 95% confidence interval, 0.94-0.99; p = 0.01). Genetically-determined resting heart rate was not associated with incident AF in the other two strata., Conclusions: For resting heart rates below 65 bpm, our results support an inverse causal association between genetically-determined resting heart rate and incident AF., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Dr. Lubitz receives sponsored research support from Bristol Myers Squibb / Pfizer, Bayer AG, Boehringer Ingelheim, Fitbit, and IBM, and has consulted for Bristol Myers Squibb / Pfizer, Bayer AG, and Blackstone Life Sciences. Dr. P.T. Ellinor is supported by a grant from Bayer AG to the Broad Institute focused on the genetics and therapeutics of cardiovascular disease, and consulted for Bayer AG, Novartis, MyoKardia, and Quest Diagnostics. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The endothelin system may play a role in the pathogenesis of vasovagal syncope (VVS) because it is implicated in blood pressure regulation. We hypothesized that endothelin-related genetic polymorphisms might modulate susceptibility to VVS. This study aimed to evaluate the possible influence of endothelin-1 (EDN1) and endothelin receptor A (EDNRA) gene variants on the occurrence of tilt-induced VVS and autonomic nervous system activity during the head-up tilt test (HUT). Results were expressed as mean +/- SEM. In 254 patients with recurrent syncope (age 45.33+/-1.22 years, 94 males, 160 females), heart rate variability (HRV) was measured during HUT. EDN1 rs5370 G>T and EDNRA rs5333 T>C gene polymorphisms were assessed using high-resolution melting analysis. There was no statistically significant association between polymorphisms EDN1 rs5370 and EDNRA rs5333 and positivity of HUT or hemodynamic types of VVS. Patients with GT or TT genotypes at the rs5370 locus of the EDN1 had significantly higher values of high-frequency (HF) and the standard deviation of the average NN intervals at the time of the syncope, and they tended to have lower low-frequency (LF) and LF/HF ratio when compared to homozygotes (GG). No statistically significant differences were found in HRV parameters concerning the EDNRA rs5333 genotypes. Our findings suggest the potential role of EDN1 rs5370 variants in regulating autonomic nervous activity and pathogenesis of VVS.

Context: Observational studies have suggested that higher resting heart rate (RHR) may be associated with increased cardiometabolic risk. However, causal associations are not fully understood., Objective: We aimed to examine the direction, strength, and causality of the associations of RHR with cardiometabolic traits., Methods: We assessed the strength of associations between measured RHR and cardiometabolic traits in 506 211 and 372 452 participants from China Kadoorie Biobank (CKB) and UK Biobank (UKB). Mendelian randomization (MR) analyses were used to make causal inferences in 99 228 and 371 508 participants from CKB and UKB, respectively., Results: We identified significant directionally concordant observational associations between RHR and higher total cholesterol, triglycerides (TG), low-density lipoprotein, C-reactive protein (CRP), glucose, body mass index, waist-hip ratio (WHR), systolic blood pressure (SBP), and diastolic blood pressure (DBP) after the Bonferroni correction. MR analyses showed that 10 beat/min higher genetically predicted RHR was trans-ethnically associated with a higher DBP (beta 2.059 [95% CI 1.544, 2.574] mmHg in CKB; 2.037 [1.845, 2.229] mmHg in UKB), higher CRP (0.180 [0.057, 0.303] log mg/L in CKB; 0.154 [0.134, 0.174] log mg/L in UKB), higher TG (0.052 [-0.009, 0.113] log mmol/L in CKB; 0.020 [0.010, 0.030] log mmol/L in UKB) and higher WHR (0.218 [-0.033, 0.469] % in CKB; 0.225 [0.111, 0.339] % in UKB). In the opposite direction, higher genetically predicted SBP, TG, glucose, and WHR, and lower high-density lipoprotein, were associated with elevated RHR., Conclusion: Our large-scale analyses provide causal evidence for associations between RHR and cardiometabolic traits, highlighting the importance of monitoring heat rate as a means of alleviating the adverse effects of metabolic disorders., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Atrial fibrillation (AF) is a morbid and heritable irregular cardiac rhythm that affects about 2%-3% of the population. Patients with early-onset AF have a strong genetic association with the disease; nonetheless, the exact underlying mechanisms need clarification. We herein present our evaluation of a 2-generation Iranian pedigree with early-onset AF. Whole-exome sequencing was applied to elucidate the genetic predisposition. Direct DNA sequencing was utilized to confirm and screen the variants in the proband and his available family members. The pathogenicity of the identified nucleotide variations was scrutinized via either segregation analysis in the family or in silico predictive software. The comprehensive variant analysis revealed a missense variant (c.G681C, p.E227D, rs1477078144) in the human α-dystrobrevin gene (DTNA), which is rare in genetic databases. Most in silico analyses have predicted this variant as a disease-causing variant, and the variant is co-segregated with the disease phenotype in the family. Previous studies have demonstrated the association between the DTNA gene and left ventricular noncompaction cardiomyopathy. Taken together, we provide the first evidence of an association between a nucleotide variation in the DTNA gene and early-onset AF in an Iranian family. However, the genetic testing of AF in the Iranian population is still limited. This finding not only further confirms the significant role of genetics in the incidence of early-onset AF but also expands the spectrum of the gene variations that lead to AF. Additionally, it may have further implications for the treatment and prevention of AF., (© 2022. The Author(s).)

Genetics crucially contributes to cardiovascular diseases (CVDs), the global leading cause of death. Since the majority of CVDs can be prevented by early intervention there is a high demand for the identification of predictive causative genes. While genome wide association studies (GWAS) correlate genes and CVDs after diagnosis and provide a valuable resource for such causative candidate genes, often preferentially those with previously known or suspected function are addressed further. To tackle the unaddressed blind spot of understudied genes, we particularly focused on the validation of human heart phenotype-associated GWAS candidates with little or no apparent connection to cardiac function. Building on the conservation of basic heart function and underlying genetics from fish to human we combined CRISPR/Cas9 genome editing of the orthologs of human GWAS candidates in isogenic medaka with automated high-throughput heart rate analysis. Our functional analyses of understudied human candidates uncovered a prominent fraction of heart rate associated genes from adult human patients impacting on the heart rate in embryonic medaka already in the injected generation. Following this pipeline, we identified 16 GWAS candidates with potential diagnostic and predictive power for human CVDs., Competing Interests: The authors have declared that no competing interests exist.

Background: Pathogenic variants of GNB5 encoding the β 5 subunit of the guanine nucleotide-binding protein cause IDDCA syndrome, an autosomal recessive neurodevelopmental disorder associated with cognitive disability and cardiac arrhythmia, particularly severe bradycardia., Methods: We used echocardiography and telemetric ECG recordings to investigate consequences of Gnb5 loss in mouse., Results: We delineated a key role of Gnb5 in heart sinus conduction and showed that Gnb5 -inhibitory signalling is essential for parasympathetic control of heart rate (HR) and maintenance of the sympathovagal balance. Gnb5 -/- mice were smaller and had a smaller heart than Gnb5 +/+ and Gnb5 +/- , but exhibited better cardiac function. Lower autonomic nervous system modulation through diminished parasympathetic control and greater sympathetic regulation resulted in a higher baseline HR in Gnb5 -/- mice. In contrast, Gnb5 -/- mice exhibited profound bradycardia on treatment with carbachol, while sympathetic modulation of the cardiac stimulation was not altered. Concordantly, transcriptome study pinpointed altered expression of genes involved in cardiac muscle contractility in atria and ventricles of knocked-out mice. Homozygous Gnb5 loss resulted in significantly higher frequencies of sinus arrhythmias. Moreover, we described 13 affected individuals, increasing the IDDCA cohort to 44 patients., Conclusions: Our data demonstrate that loss of negative regulation of the inhibitory G-protein signalling causes HR perturbations in Gnb5 - /- mice, an effect mainly driven by impaired parasympathetic activity. We anticipate that unravelling the mechanism of Gnb5 signalling in the autonomic control of the heart will pave the way for future drug screening., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)

Background: The association between resting heart rate (HR) and cardiovascular outcomes, especially heart failure, is now well established. However, whether HR is mainly an integrated marker of risk associated with other features, or rather a genetic origin risk marker, is still a matter for debate. Previous studies reported a heritability ranging from 14% to 65%., Design: We assessed HR heritability in the STANISLAS family-study, based on the data of four visits performed over a 20-year period, and adjusted for most known confounding effects., Methods: These analyses were conducted using a linear mixed model, adjusted on age, sex, tea or coffee consumption, beta-blocker use, physical activity, tobacco use, and alcohol consumption to estimate the variance captured by additive genetic effects, via average information restricted maximum likelihood analysis, with both self-reported pedigree and genetic relatedness matrix (GRM) calculated from genome-wide association study data., Results: Based on the data of all visits, the HR heritability (h2) estimate was 23.2% with GRM and 24.5% with pedigree. However, we found a large heterogeneity of HR heritability estimations when restricting the analysis to each of the four visits (h2 from 19% to 39% using pedigree, and from 14% to 32% using GRM). Moreover, only a little part of variance was explained by the common household effect (<5%), and half of the variance remained unexplained., Conclusion: Using a comprehensive analysis based on a family cohort, including the data of multiple visits and GRM, we found that HR variability is about 25% from genetic origin, 25% from repeated measures and 50% remains unexplained., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

Sickness behaviors, including anorexia, are evolutionarily conserved responses to acute infections. Inflammation-induced anorexia causes dramatic metabolic changes, of which components critical to survival are unique depending on the type of inflammation. Glucose supplementation during the anorectic period induced by bacterial inflammation suppresses adaptive fasting metabolic pathways, including fibroblast growth factor 21 (FGF21), and decreases survival. Consistent with this observation, FGF21-deficient mice are more susceptible to mortality from endotoxemia and polybacterial peritonitis. Here, we report that increased circulating FGF21 during bacterial inflammation is hepatic derived and required for survival through the maintenance of thermogenesis, energy expenditure, and cardiac function. FGF21 signaling downstream of its obligate coreceptor, β-Klotho (KLB), is required in bacterial sepsis. However, FGF21 modulates thermogenesis and chronotropy independent of the adipose, forebrain, and hypothalamus, which are operative in cold adaptation, suggesting that in bacterial inflammation, either FGF21 signals through a novel, undescribed target tissue or concurrent signaling of multiple KLB-expressing tissues is required., Competing Interests: Disclosures: H.H. Luan reported "other" from NGM Biopharmaceuticals outside the submitted work. No other disclosures were reported., (© 2021 Huen et al.)

Background and Aims: Observational studies have associated resting heart rate with incident diabetes. Whether the associations are causal remains unclear. We aimed to examine the shape and strength of the associations and assessed the causal relevance of such associations in Chinese adults., Methods and Results: The China Kadoorie Biobank enrolled 512,891 adults in China. Cox proportional hazard regression models was conducted to estimate hazard ratios (HRs) for the associations of resting heart rate with type 2 diabetes and total diabetes. Among 92,724 participants, 36 single-nucleotide polymorphisms (SNPs) related to resting heart rate were used to construct genetic risk score. We used Mendelian randomization analyses to make the causal inferences. During a median follow-up of 9 years, 7872 incident type 2 diabetes and 13,349 incident total diabetes were documented. After regression dilution bias adjustment, each 10 bpm higher heart rate was associated with about a 26% higher risk of type 2 diabetes (HR, 1.26 [95% CI, 1.23, 1.29]) and 23% higher risk of total diabetes (HR, 1.23 [95% CI, 1.20, 1.26]). Instrumental variable analyses showed participants at top quintile compared with those at bottom quintile had 30% higher risk for type 2 diabetes (HR, 1.30 [95% CI, 1.17, 1.43]), and 10% higher risk for total diabetes (HR, 1.10 [95% CI, 1.02, 1.20])., Conclusions: This study provides evidence that resting heart rate is an important risk factor for diabetes risk. The results suggest that novel treatment approaches targeting reduction of high heart rate for incidence of diabetes may be worth further investigation., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2021 The Italian Diabetes Society, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.)

The preoptic area (POA) is a key brain region for regulation of body temperature (Tb), dictating thermogenic, cardiovascular, and behavioral responses that control Tb. Previously characterized POA neuronal populations all reduced Tb when activated. Using mice, we now identify POA neurons expressing bombesin-like receptor 3 (POA BRS3 ) as a population whose activation increased Tb; inversely, acute inhibition of these neurons reduced Tb. POA BRS3 neurons that project to either the paraventricular nucleus of the hypothalamus or the dorsomedial hypothalamus increased Tb, heart rate, and blood pressure via the sympathetic nervous system. Long-term inactivation of POA BRS3 neurons caused increased Tb variability, overshooting both increases and decreases in Tb set point, with RNA expression profiles suggesting multiple types of POA BRS3 neurons. Thus, POA BRS3 neuronal populations regulate Tb and heart rate, contribute to cold defense, and fine-tune feedback control of Tb. These findings advance understanding of homeothermy, a defining feature of mammalian biology., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Endothelial cells (ECs) secrete different paracrine signals that modulate the function of adjacent cells; two examples of these paracrine signals are nitric oxide (NO) and neuregulin-1 (NRG1), a cardioprotective growth factor. Currently, it is undetermined whether one paracrine factor can compensate for the loss of another. Herein, we hypothesized that NRG1 can compensate for endothelial NO synthase (eNOS) deficiency. We characterized eNOS null and wild-type (WT) mice by cardiac ultrasound and histology and we determined circulating NRG1 levels. In a separate experiment, eight groups of mice were divided into four groups of eNOS null mice and WT mice; half of the mice received angiotensin II (ANG II) to induce a more severe phenotype. Mice were randomized to daily injections with NRG1 or vehicle for 28 days. eNOS deficiency increased NRG1 plasma levels, indicating that ECs increase their NRG1 expression when NO production is deleted. eNOS deficiency also increased blood pressure, lowered heart rate, induced cardiac fibrosis, and affected diastolic function. In eNOS null mice, ANG II administration not only increased cardiac fibrosis but also induced cardiac hypertrophy and renal fibrosis. NRG1 administration prevented cardiac and renal hypertrophy and fibrosis caused by ANG II infusion and eNOS deficiency. Moreover, Nrg1 expression in the myocardium is shown to be regulated by miR-134. This study indicates that administration of endothelium-derived NRG1 can compensate for eNOS deficiency in the heart and kidneys. NEW & NOTEWORTHY ECs compensate for eNOS deficiency by increasing the secretion of NRG1. NRG1 administration prevents cardiac and renal hypertrophy and fibrosis caused by ANG II infusion and eNOS deficiency. NRG1 expression is regulated by miR-134.

The establishment of cardiac function in the developing embryo is essential to ensure blood flow and, therefore, growth and survival of the animal. The molecular mechanisms controlling normal cardiac rhythm remain to be fully elucidated. From a forward genetic screen, we identified a unique mutant, grime, that displayed a specific cardiac arrhythmia phenotype. We show that loss-of-function mutations in tmem161b are responsible for the phenotype, identifying Tmem161b as a regulator of cardiac rhythm in zebrafish. To examine the evolutionary conservation of this function, we generated knockout mice for Tmem161b. Tmem161b knockout mice are neonatal lethal and cardiomyocytes exhibit arrhythmic calcium oscillations. Mechanistically, we find that Tmem161b is expressed at the cell membrane of excitable cells and live imaging shows it is required for action potential repolarization in the developing heart. Electrophysiology on isolated cardiomyocytes demonstrates that Tmem161b is essential to inhibit Ca 2+ and K + currents in cardiomyocytes. Importantly, Tmem161b haploinsufficiency leads to cardiac rhythm phenotypes, implicating it as a candidate gene in heritable cardiac arrhythmia. Overall, these data describe Tmem161b as a highly conserved regulator of cardiac rhythm that functions to modulate ion channel activity in zebrafish and mice., Competing Interests: The authors declare no competing interest.

Vagus nerve stimulation has shown many benefits for disease therapies but current approaches involve imprecise electrical stimulation that gives rise to off-target effects, while the functionally relevant pathways remain poorly understood. One method to overcome these limitations is the use of optogenetic techniques, which facilitate targeted neural communication with light-sensitive actuators (opsins) and can be targeted to organs of interest based on the location of viral delivery. Here, we tested whether retrograde adeno-associated virus (rAAV2-retro) injected in the heart can be used to selectively express opsins in vagus nerve fibers controlling cardiac function. Furthermore, we investigated whether perturbations in cardiac function could be achieved with photostimulation at the cervical vagus nerve. Viral injection in the heart resulted in robust, primarily afferent, opsin reporter expression in the vagus nerve, nodose ganglion, and brainstem. Photostimulation using both one-photon stimulation and two-photon holography with a GRIN-lens incorporated nerve cuff, was tested on the pilot-cohort of injected mice. Changes in heart rate, surface electrocardiogram, and respiratory responses were observed in response to both one- and two-photon photostimulation. The results demonstrate feasibility of retrograde labeling for organ targeted optical neuromodulation.

Physiological systems vary in a day-night manner anticipating increased demand at a particular time. Heart is no exception. Cardiac output is primarily determined by heart rate and unsurprisingly this varies in a day-night manner and is higher during the day in the human (anticipating increased day-time demand). Although this is attributed to a day-night rhythm in post-translational ion channel regulation in the heart's pacemaker, the sinus node, by the autonomic nervous system, we investigated whether there is a day-night rhythm in transcription. RNAseq revealed that ~ 44% of the sinus node transcriptome (7134 of 16,387 transcripts) has a significant day-night rhythm. The data revealed the oscillating components of an intrinsic circadian clock. Presumably this clock (or perhaps the master circadian clock in the suprachiasmatic nucleus) is responsible for the rhythm observed in the transcriptional machinery, which in turn is responsible for the rhythm observed in the transcriptome. For example, there is a rhythm in transcripts responsible for the two principal pacemaker mechanisms (membrane and Ca 2+ clocks), transcripts responsible for receptors and signalling pathways known to control pacemaking, transcripts from genes identified by GWAS as determinants of resting heart rate, and transcripts from genes responsible for familial and acquired sick sinus syndrome.

Background: Studies indicate that chronic vitamin D deficiency (VDD) may predispose to hypertension, yet, there is very little data characterizing its direct cardiac effects. Vitamin D modulates the function of transient receptor potential C cation channels (TRPC), which is a mechanosensitive cation channel that plays a role in cardiac slow-force responses to hemodynamic changes. The purpose of this study was to determine the cardiac effects of VDD and the potential role of TRPC., Methods: Three-week old mice were placed on a VDD or normal diet (ND) for 19 weeks. Mice were then implanted with radiotelemeters for the measurement of heart rate (HR) and heart rate variability (HRV), while a separate group was anesthetized to measure blood pressure (BP) and left ventricular function using an intraventricular probe. Animals were treated with a TRPC antagonist or vehicle after which they were challenged with dobutamine to measure cardiac responses., Results: VDD mice had significantly increased BP (72 ± 3 mmHg vs. 62 ± 2 mmHg) and left ventricular pressure (LVP) (84.6 ± 0.8 mmHg vs. 78.2 ± 2.0 mmHg), and decreased cardiac contractility (-3 % vs. + 11 %) and HR response (+8 % vs. + 13 %) to dobutamine when compared to ND. These responses were blocked by the TRPC antagonist. HRV decreased with increasing dobutamine doses in ND but not VDD mice, however, the antagonist had no effect., Conclusion: VDD increases BP and alters cardiac mechanical function in mice, the latter appears to be mediated by TRPC, in particular TRPC6. Although the cardiac effects might be due to increased BP, it is likely that VDD also affects the function of the heart directly. This is the first study to demonstrate the potentially deleterious effects of VDD on cardiac function and the role of TRPC6 in this response., (Published by Elsevier Ltd.)

This study aims to analyze the behaviour of cardiac autonomic modulation in adolescents with a family history of hypertension along with polymorphism of the ACE gene (rs1799752). The study involved 141 adolescents, with a mean age of 14.89, divided into the following six groups: offspring of normotensive parents (ONP): DD, DI and II; and offspring of hypertensive parents (OHP): DD, DI and II. Blood pressure, body composition, family history of hypertension, sleep disorder, and sexual maturation were assessed for the groups' characterization. Afterwards, an electrocardiogram was performed, and oral mucosal cells were collected to analyze heart rate variability and genotypic research of angiotensin-converting enzyme. The main finding of this study was the decrease of vagal action in group OHP (genotype DD) relative to group ONP (genotype II): LF (%), 54.25 ± 3.14 vs 39.33 ± 3.80; HF (%), 45.74 ± 3.14 vs 60.66 ± 3.80; LF/HF, 1.48 ± 0.23 vs 0.68 ± 0.19. The results also showed changes in the variable diastolic blood pressure (DBP) in OHP (genotype DI) to ONP (genotype II) groups: 72.99 ± 2.33 vs 63.27 ± 1.72; and OHP (genotype DI) to ONP (genotype DD) groups. Adolescents with genotype DD and a family history of arterial hypertension present chances in cardiac autonomic modulation, the cardiac parasympathetic modulation is lower in these adolescents in comparison to participants of ONP + II group., (© 2020 John Wiley & Sons Australia, Ltd.)

Reduced expression of the plasma membrane citrate transporter INDY (acronym I'm Not Dead, Yet) extends life span in lower organisms. Deletion of the mammalian Indy (mIndy) gene in rodents improves metabolism via mechanisms akin to caloric restriction, known to lower blood pressure (BP) by sympathoadrenal inhibition. We hypothesized that mIndy deletion attenuates sympathoadrenal support of BP. Continuous arterial BP and heart rate (HR) were reduced in mINDY-KO mice. Concomitantly, urinary catecholamine content was lower, and the decreases in BP and HR by mIndy deletion were attenuated after autonomic ganglionic blockade. Catecholamine biosynthesis pathways were reduced in mINDY-KO adrenals using unbiased microarray analysis. Citrate, the main mINDY substrate, increased catecholamine content in pheochromocytoma cells, while pharmacological inhibition of citrate uptake blunted the effect. Our data suggest that deletion of mIndy reduces sympathoadrenal support of BP and HR by attenuating catecholamine biosynthesis. Deletion of mIndy recapitulates beneficial cardiovascular and metabolic responses to caloric restriction, making it an attractive therapeutic target.

Disparities between the races have been well documented in health and disease in the USA. Recent studies show that telomere length, a marker of aging, is associated with obesity and obesity-related diseases, such as heart disease and diabetes. The current study aimed to evaluate the connection between telomere length ratio, blood pressure, and childhood obesity. The telomere length ratio was measured in 127 children from both European American (EA) and African American (AA) children, aged 6-10 years old. AA children had a significantly high relative telomere to the single copy gene (T/S) ratio compared to EA children. There was no significant difference in the T/S ratio between normal weight (NW) and overweight/obese (OW/OB) groups of either race. Blood pressure was significantly elevated in AA children with respect to EA children. Hierarchical regression analysis adjusted for race, gender, and age expressed a significant relationship between the T/S ratio and diastolic pressure. Low T/S ratio participants showed a significant increase in systolic pressure, while a high T/S ratio group showed an increase in diastolic pressure and heart rate of AA children. In conclusion, our findings show that AA children have high T/S ratio compared to EA children. The high T/S ratio is negatively associated with diastolic pressure.

Optogenetics is a new approach using light intensity to modulate the electrical activity of excitable cells by the interaction of light-sensitive proteins. This method has been widely and enthusiastically utilized in some fields over the last decade. Localizing a photosensitive protein to a specific place in the membrane of cardiomyocytes at a specific time is essential for most biological processes. In this case, vectors are injected into the circulation to allow them to spread throughout the whole heart. The aim of this protocol is to perform different illumination modes with blue laser to investigate optical control of Langendorff-perfused mice hearts which were systematically injected with adeno-associated virus (AAV) for ChR2(H134R) gene transfer. Electrograms (EGs) and epicardium monophasic action potential (MAP) showed that ChR2 expression in the heart can be flexibly controlled by blue light across different illumination sites with corresponding triggered ectopic rhythm. Illumination intensity, pulse duration, and impulse frequency were associated with the light capture rate. Flexible control of the cardiac rhythm with optogenetics provides an innovative approach to cardiac research and therapy.

ATP is a major energy source in the mammalian cells, but it is an extracellular chemical messenger acting on P2 purinergic receptors. A line of evidence has shown that ATP is released from many different types of cells including neurons, endothelial cells, and muscle cells. In this review, we described the distribution of P2 receptor subtypes in the cardiac cells and their physiological and pathological roles in the heart. So far, the effects of external application of ATP or its analogues, and those of UTP on cardiac contractility and rhythm have been reported. In addition, specific genetic alterations and pharmacological agonists and antagonists have been adopted to discover specific roles of P2 receptor subtypes including P2X4-, P2X7-, P2Y2- and P2Y6-receptors in cardiac cells under physiological and pathological conditions. Accumulated data suggest that P2X4 receptors may play a beneficial role in cardiac muscle function, and that P2Y2- and P2Y6-receptors can induce cardiac fibrosis. Recent evidence further demonstrates P2Y1 receptor and P2X4 receptor as important mechanical signaling molecules to alter membrane potential and Ca 2+ signaling in atrial myocytes and their uneven expression profile between right and left atrium.

Background: The diagnosis of long QT syndrome (LQTS) is rather straightforward. We were surprised by realizing that, despite long-standing experience, we were making occasional diagnostic errors by considering as affected subjects who, over time, resulted as not affected. These individuals were all actively practicing sports-an observation that helped in the design of our study., Methods: We focused on subjects referred to our center by sports medicine doctors on suspicion of LQTS because of marked repolarization abnormalities on the ECG performed during the mandatory medical visit necessary in Italy to obtain the certificate of eligibility to practice sports. They all underwent our standard procedures involving both a resting and 12-lead ambulatory ECG, an exercise stress test, and genetic screening., Results: There were 310 such consecutive subjects, all actively practicing sports with many hours of intensive weekly training. Of them, 111 had a normal ECG, different cardiac diseases, or were lost to follow-up and exited the study. Of the remaining 199, all with either clear QTc prolongation and/or typical repolarization abnormalities, 121 were diagnosed as affected based on combination of ECG abnormalities with positive genotyping (QTc, 482±35 ms). Genetic testing was negative in 78 subjects, but 45 were nonetheless diagnosed as affected by LQTS based on unequivocal ECG abnormalities (QTc, 472±33 ms). The remaining 33, entirely asymptomatic and with a negative family history, showed an unexpected and practically complete normalization of the ECG abnormalities (their QTc shortened from 492±37 to 423±25 ms [ P <0.001]; their Schwartz score went from 3.0 to 0.06) after detraining. They were considered not affected by congenital LQTS and are henceforth referred to as "cases." Furthermore, among them, those who resumed similarly heavy physical training showed reappearance of the repolarization abnormalities., Conclusion: It is not uncommon to suspect LQTS among individuals actively practicing sports based on marked repolarization abnormalities. Among those who are genotype-negative, >40% normalize their ECG after detraining, but the abnormalities tend to recur with resumption of training. These individuals are not affected by congenital LQTS but could have a form of acquired LQTS. Care should be exercised to avoid diagnostic errors.

The Transient Receptor Potential Melastatin 4 (TRPM4) is a transmembrane N-glycosylated ion channel that belongs to the large family of TRP proteins. It has an equal permeability to Na + and K + and is activated via an increase of the intracellular calcium concentration and membrane depolarization. Due to its wide distribution, TRPM4 dysfunction has been linked with several pathophysiological processes, including inherited cardiac arrhythmias. Many pathogenic variants of the TRPM4 gene have been identified in patients with different forms of cardiac disorders such as conduction defects, Brugada syndrome, and congenital long QT syndrome. At the cellular level, these variants induce either gain- or loss-of-function of TRPM4 channels for similar clinical phenotypes. However, the molecular mechanisms associating these functional alterations to the clinical phenotypes remain poorly understood. The main objective of this article is to review the major cardiac TRPM4 channelopathies and recent advances regarding their genetic background and the underlying molecular mechanisms., Competing Interests: Prof. Mohamed Yassine Amarouch declares that he has no conflict of interest. Prof. Jaouad El Hilaly declares that he has no conflict of interest., (Copyright © 2020 Mohamed-Yassine Amarouch and Jaouad El Hilaly.)

Cardiac ischemia is associated with arrhythmias; however, effective therapies are currently limited. The cardiac voltage-gated sodium channel α subunit (SCN5A), encoding the Nav1.5 current, plays a key role in the cardiac electrical conduction and arrhythmic risk. Here, we show that hypoxia reduces Nav1.5 through effects on a miR, miR-448. miR-448 expression is increased in ischemic cardiomyopathy. miR-448 has a conserved binding site in 3'-UTR of SCN5A. miR-448 binding to this site suppressed SCN5A expression and sodium currents. Hypoxia-induced HIF-1α and NF-κB were major transcriptional regulators for MIR448. Moreover, hypoxia relieved MIR448 transcriptional suppression by RE1 silencing transcription factor. Therefore, miR-448 inhibition reduced arrhythmic risk after myocardial infarction. Here, we show that ischemia drove miR-448 expression, reduced Nav1.5 current, and increased arrhythmic risk. Arrhythmic risk was improved by preventing Nav1.5 downregulation, suggesting a new approach to antiarrhythmic therapy.

Autonomic nervous function evaluated by heart rate variability (HRV) and blood characteristics were compared between Holstein Friesian cows that developed postpartum fever (PF; n = 5) and clinically healthy (CH; n = 6) puerperal cows in this case-control study. A cow was defined as having PF when its rectal temperature rose to ≥39.5°C between 1 and 3 days postpartum. We recorded electrocardiograms during this period using a Holter-type electrocardiograph and applied power spectral analysis of HRV. Comparisons between the groups were analyzed by t test or Mann-Whitney U test, and the relationship between rectal temperature and each parameter was analyzed using multiple regression analysis. Heart rate was higher in PF cows than in CH cows (Mean ± SE, 103.3 ± 2.7 vs. 91.5 ± 1.7 bpm). This result suggested that PF cows had a relatively dominant sympathetic nervous function. Total (44,472 ± 2,301 vs. 55,373 ± 1,997 ms) and low frequency power (24.5 ± 3.8 vs. 39.9 ± 5.3 ms) were lower in PF cows than in CH cows. These findings were possibly caused by a reduction in autonomic nervous function. The total white blood cell count (54.3 ± 5.1 vs. 84.5 ± 6.4 ×102/μL) and the serum magnesium (2.1 ± 0.1 vs. 2.4 ± 0.1 mg/dL) and iron (81.5 ± 8.0 vs. 134.4 ± 9.1 μg/dL) concentrations were lower and the serum amyloid A concentration (277 ± 33 vs. 149 ± 21 μg/mL) was higher in PF cows than in CH cows. These results imply that more inflammation was present in PF cows than in CH cows. Multiple regression analysis showed that both of low frequency power and concentration of serum iron were associated with rectal temperature. We found differences in changes in hematologic results, biochemical findings, and HRV patterns between PF cows and CH cows., Competing Interests: The authors have declared that no competing interests exist.

Aims: The genetic cause of cardiac conduction system disease (CCSD) has not been fully elucidated. Whole-exome sequencing (WES) can detect various genetic variants; however, the identification of pathogenic variants remains a challenge. We aimed to identify pathogenic or likely pathogenic variants in CCSD patients by using WES and 2015 American College of Medical Genetics and Genomics (ACMG) standards and guidelines as well as evaluating the usefulness of functional studies for determining them., Methods and Results: We performed WES of 23 probands diagnosed with early-onset (<65 years) CCSD and analysed 117 genes linked to arrhythmogenic diseases or cardiomyopathies. We focused on rare variants (minor allele frequency < 0.1%) that were absent from population databases. Five probands had protein truncating variants in EMD and LMNA which were classified as 'pathogenic' by 2015 ACMG standards and guidelines. To evaluate the functional changes brought about by these variants, we generated a knock-out zebrafish with CRISPR-mediated insertions or deletions of the EMD or LMNA homologs in zebrafish. The mean heart rate and conduction velocities in the CRISPR/Cas9-injected embryos and F2 generation embryos with homozygous deletions were significantly decreased. Twenty-one variants of uncertain significance were identified in 11 probands. Cellular electrophysiological study and in vivo zebrafish cardiac assay showed that two variants in KCNH2 and SCN5A, four variants in SCN10A, and one variant in MYH6 damaged each gene, which resulted in the change of the clinical significance of them from 'Uncertain significance' to 'Likely pathogenic' in six probands., Conclusion: Of 23 CCSD probands, we successfully identified pathogenic or likely pathogenic variants in 11 probands (48%). Functional analyses of a cellular electrophysiological study and in vivo zebrafish cardiac assay might be useful for determining the pathogenicity of rare variants in patients with CCSD. SCN10A may be one of the major genes responsible for CCSD., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: journals.permissions@oup.com.)

Background The sinus node (SN) is the primary pacemaker of the heart. SN myocytes possess distinctive action potential morphology with spontaneous diastolic depolarization because of a unique expression of ion channels and Ca 2+ -handling proteins. MicroRNAs (miRs) inhibit gene expression. The role of miRs in controlling the expression of genes responsible for human SN pacemaking and conduction has not been explored. The aim of this study was to determine miR expression profile of the human SN as compared with that of non-pacemaker atrial muscle. Methods and Results SN and atrial muscle biopsies were obtained from donor or post-mortem hearts (n=10), histology/immunolabeling were used to characterize the tissues, TaqMan Human MicroRNA Arrays were used to measure 754 miRs, Ingenuity Pathway Analysis was used to identify miRs controlling SN pacemaker gene expression. Eighteen miRs were significantly more and 48 significantly less abundant in the SN than atrial muscle. The most interesting miR was miR-486-3p predicted to inhibit expression of pacemaking channels: HCN1 (hyperpolarization-activated cyclic nucleotide-gated 1), HCN4, voltage-gated calcium channel (Ca v )1.3, and Ca v 3.1. A luciferase reporter gene assay confirmed that miR-486-3p can control HCN4 expression via its 3' untranslated region. In ex vivo SN preparations, transfection with miR-486-3p reduced the beating rate by ≈35±5% ( P <0.05) and HCN4 expression ( P <0.05). Conclusions The human SN possesses a unique pattern of expression of miRs predicted to target functionally important genes. miR-486-3p has an important role in SN pacemaker activity by targeting HCN4, making it a potential target for therapeutic treatment of SN disease such as sinus tachycardia.

Dysregulation of the cardiac autonomic nervous system, as indexed by reduced heart rate variability (HRV), has been associated with the development of high blood pressure (BP). However, the underlying pathological mechanisms are not yet fully understood. This study aimed to estimate heritability of HRV and BP and to determine their genetic overlap. We used baseline data of the 3-generation Lifelines population-based cohort study (n=149 067; mean age, 44.5). In-house software was used to calculate root mean square of successive differences and SD of normal-to-normal intervals as indices of HRV based on 10-second resting ECGs. BP was recorded with an automatic BP monitor. We estimated heritabilities and genetic correlations with variance components methods in ASReml software. We additionally estimated genetic correlations with bivariate linkage disequilibrium score regression using publicly available genome-wide association study data. The heritability (SE) estimates were 15.6% (0.90%) for SD of normal-to-normal intervals and 17.9% (0.90%) for root mean square of successive differences. For BP measures, they ranged from 24.4% (0.90%) for pulse pressure to 30.3% (0.90%) for diastolic BP. Significant negative genetic correlations (all P <0.0001) of root mean square of successive differences/SD of normal-to-normal intervals with systolic BP (-0.20/-0.16) and with diastolic BP (-0.15/-0.13) were observed. LD score regression showed largely consistent genetic correlation estimates of root mean square of successive differences/SD of normal-to-normal intervals with systolic BP (range, -0.08 to -0.23) and diastolic BP (range, -0.20 to -0.27). Our study shows a substantial contribution of genetic factors in explaining the variance of HRV and BP measures in the general population. The significant negative genetic correlations between HRV and BP indicate that genetic pathways for HRV and BP partially overlap.

Background: In cardiac gene therapy to improve contractile function, achieving gene expression in the majority of cardiac myocytes is essential. In preventing cardiac arrhythmias, however, this goal may not be as important since transduction efficiencies as low as 40% suppressed ventricular arrhythmias in genetically modified mice with catecholaminergic polymorphic ventricular tachycardia., Methods: Using computational modeling, we simulated 1-, 2-, and 3-dimensional tissue under a variety of conditions to test the ability of genetically engineered nonarrhythmogenic stabilizer cells to suppress triggered activity due to delayed or early afterdepolarizations., Results: Due to source-sink relationships in cardiac tissue, a minority (20%-50%) of randomly distributed stabilizer cells engineered to be nonarrhythmogenic can suppress the ability of arrhythmogenic cells to generate delayed and early afterdepolarizations-related arrhythmias. Stabilizer cell gene therapy strategy can be designed to correct a specific arrhythmogenic mutation, as in the catecholaminergic polymorphic ventricular tachycardia mice studies, or more generally to suppress delayed or early afterdepolarizations from any cause by overexpressing the inward rectifier K channel Kir2.1 in stabilizer cells., Conclusions: This promising antiarrhythmic strategy warrants further testing in experimental models to evaluate its clinical potential.

Cardiac sympathetic innervation is critically involved in the regulation of circulatory dynamics. However, the molecular mechanism for the innervation patterning has remained elusive. Here, we demonstrate that nardilysin (NRDC, Nrdc), an enhancer of ectodomain shedding, regulates cardiac sympathetic innervation. Nardilysin-deficient (Nrdc -/- ) mice show hypoplastic hearts, hypotension, bradycardia, and abnormal sympathetic innervation patterning. While the innervation of left ventricle (LV) of wild-type mice is denser in the subepicardium than in the subendocardium, Nrdc -/- LV lacks such a polarity and is uniformly and more abundantly innervated. At the molecular level, the full-length form of p75 neurotrophin receptor (p75 NTR , Ngfr) is increased in Nrdc -/- LV due to the reduced ectodomain shedding of p75 NTR . Importantly, the reduction of p75 NTR rescued the abnormal innervation phenotype of Nrdc -/- mice. Moreover, sympathetic neuron-specific, but not cardiomyocyte-specific deletion of Nrdc recapitulated the abnormal innervation patterning of Nrdc -/- mice. In conclusion, neuronal nardilysin critically regulates cardiac sympathetic innervation and circulatory dynamics via modulation of p75 NTR ., (© 2020 Federation of American Societies for Experimental Biology.)

Background: Atrial fibrillation (AF) is one of the most common arrhythmia, which brings huge burden to the individual and the society. However, the mechanism of AF is not clear. This paper aims at screening the key differentially expressed genes (DEGs) of atrial fibrillation and to construct enrichment analysis and protein-protein interaction (PPI) network analysis for these DEGs., Methods: The datasets were collected from the Gene Expression Omnibus database to extract data of left atrial appendage (LAA) RNA of patients with or without AF in GSE79768, GSE31821, GSE115574, GSE14975 and GSE41177. Batch normalization, screening of the differential genes and gene ontology analysis were finished by R software. Reactome analysis was used for pathway analysis. STRING platform was utilized for PPI network analysis. At last, we performed reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to validate the expression of key genes in 20 sinus rhythm (SR) LAA tissues and 20 AF LAA tissues., Results: A total of 106 DEGs were screened in the merged dataset. Among these DEGs, 74 genes were up-regulated and 32 genes down-regulated. DEGs were mostly enriched in extracellular matrix organization, protein activation cascade and extracellular structure organization. In PPI network, we identified SPP1, COL5A1 and VCAN as key genes which were associated with extracellular matrix. RT-qPCR showed the same expression trend of the three key genes as in our bioinformatics analysis. The expression levels of SPP1, COL5A1 and VCAN were increased in AF tissues compared to SR tissues (P < 0.05)., Conclusion: According to the analyses which were conducted by bioinformatics tools, genes related to extracellular matrix were involved in pathology of AF and may become the possible targets for the diagnosis and treatment of AF.

Graves' disease is an autoimmune disorder, which is characterized by stimulatory antibodies targeting the human thyrotropin receptor (TSHR), resulting in hyperthyroidism and multiple organ damage. We systematically investigated monomeric and dimeric fusion proteins of the A subunit of TSHR for efficacy to bind to the monoclonal patient antibody M22, to interact with Graves' patient serum samples, and to impact on anti-TSHR antibody titers, hyperthyroidism, tachycardia and other in vivo read-outs in a long-term mouse model of Graves' disease induced by immunization with a recombinant adenovirus encoding TSHR A. Binding assays and functional measurements of TSHR-dependent cAMP formation showed binding of monomeric TSHR-His and dimeric TSHR-Fc to the anti-TSHR antibody M22 at low-effective concentrations (EC50 of 5.7 nmol/L and 8.6 nmol/L) and inhibition of the effects of this antibody at high efficiencies (IC50 values of 16-20 nmol/L). Both proteins also block the effects of polyclonal anti-TSHR antibodies occurring in Graves' patient sera with somewhat lower average efficiencies (mean IC50 values of 29 nmol/L and 68 nmol/L). However, in vivo characterization of epicutaneous patch administrations of TSHR-Fc at doses of 0.3 and 0.6 mg/kg body weight in a murine Graves' disease model did not result in any improvement of disease parameters. In conclusion, high affinity binding of TSHR-Fc to pathological anti-TSHR antibodies was not matched by efficacy to improve Graves' disease parameter in a long-term mouse model., (© 2020 Society for Endocrinology.)

Background: Naturally occurring human genetic variants provide a valuable tool to identify drug targets and guide drug prioritization and clinical trial design. Ivabradine is a heart rate lowering drug with protective effects on heart failure despite increasing the risk of atrial fibrillation. In patients with coronary artery disease without heart failure, the drug does not protect against major cardiovascular adverse events prompting questions about the ability of genetics to have predicted those effects. This study evaluates the effect of a variant in HCN4, ivabradine's drug target, on safety and efficacy endpoints., Methods: We used genetic association testing and Mendelian randomization to predict the effect of ivabradine and heart rate lowering on cardiovascular outcomes., Results: Using data from the UK Biobank and large GWAS consortia, we evaluated the effect of a heart rate-reducing genetic variant at the HCN4 locus encoding ivabradine's drug target. These genetic association analyses showed increases in risk for atrial fibrillation (OR 1.09, 95% CI: 1.06-1.13, P = 9.3 ×10-9) in the UK Biobank. In a cause-specific competing risk model to account for the increased risk of atrial fibrillation, the HCN4 variant reduced incident heart failure in participants that did not develop atrial fibrillation (HR 0.90, 95% CI: 0.83-0.98, P = 0.013). In contrast, the same heart rate reducing HCN4 variant did not prevent a composite endpoint of myocardial infarction or cardiovascular death (OR 0.99, 95% CI: 0.93-1.04, P = 0.61)., Conclusion: Genetic modelling of ivabradine recapitulates its benefits in heart failure, promotion of atrial fibrillation, and neutral effect on myocardial infarction., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: A patent pertaining to pharmacogenomics-guided CETP inhibition (US20190070178A1) owned by Dalcor was granted and J.-C. Tardif and M.-P. Dubé are mentioned as authors but do not receive any royalties. J.-C. Tardif and M.-P. Dubé have a minor equity interest in DalCor. J.-C. Tardif has received research support from Amarin, AstraZeneca, DalCor, Eli-Lilly, Ionis, Pfizer, RegenexBio, Sanofi and Servier, and honoraria from DalCor, Pfizer, Sanofi and Servier. M.-P. Dubé has received honoraria from Dalcor and research support (access to samples and data) from AstraZeneca, Pfizer, Servier, Sanofi and GlaxoSmithKline. B. Tyl is an employee of Laboratoires Servier. Simon de Denus has received grants from Pfizer, AstraZeneca, Roche Molecular Science, DalCor and Novartis. R. Thomas Lumbers has received research grants from Pfizer. The remaining authors have nothing to disclose. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Inherited arrhythmias are disorders caused by one or more genetic mutations that increase the risk of arrhythmia, which result in life-long risk of sudden death. These mutations either primarily perturb electrophysiological homeostasis (e.g. long QT syndrome and catecholaminergic polymorphic ventricular tachycardia), cause structural disease that is closely associated with severe arrhythmias (e.g. hypertrophic cardiomyopathy), or cause a high propensity for arrhythmia in combination with altered myocardial structure and function (e.g. arrhythmogenic cardiomyopathy). Currently available therapies offer incomplete protection from arrhythmia and fail to alter disease progression. Recent studies suggest that gene therapies may provide potent, molecularly targeted options for at least a subset of inherited arrhythmias. Here, we provide an overview of gene therapy strategies, and review recent studies on gene therapies for catecholaminergic polymorphic ventricular tachycardia and hypertrophic cardiomyopathy caused by MYBPC3 mutations., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: journals.permissions@oup.com.)