Heart rate (HR) response to exercise and recovery are strong predictors of cardiovascular mortality, but the HR profile morphology may add useful information for the identification of subjects at risk.Our aim was to characterise the HR profile morphology and assess its cardiovascular risk predictive value.1-lead ECG recordings of 17,691 participants from the general population in an exercise stress test from the UK Biobank study were analyzed.A methodology based on time warping of the HR profile was applied to compute the average HR profile morphology along the exercise test.Then, two series of warping-based morphological differences in amplitude, d a , and time, d w , were calculated by comparing each individual HR profile morphology with respect to this average HR profile.Subjects who suffered one or more cardiovascular events showed significantly lower values of d a than survivors (median of -10.5% vs -7.5%, p=0.009).Also, d a was significantly associated with cardiovascular mortality in a Cox model after adjusting for clinical variables, resting HR, difference between peak and resting HR, or between peak and recovery HR after a follow-up period of five years (p<0.0001).Individuals at risk show HR dynamics with slower adaptation to exercise than healthier subjects, possibly due to autonomic nervous system dysfunction.
Regulators of G-protein signaling (RGS) proteins modulate signaling through heterotrimeric G-proteins. They act to enhance the intrinsic GTPase activity of the Galpha subunit but paradoxically have also been shown to enhance receptor-stimulated activation. To study this paradox, we used a G-protein gated K+ channel to report the dynamics of the G-protein cycle and fluorescence resonance energy transfer techniques with cyan and yellow fluorescent protein-tagged proteins to report physical interaction. Our data show that the acceleration of the activation kinetics is dissociated from deactivation kinetics and dependent on receptor and RGS type, G-protein isoform, and RGS expression levels. By using fluorescently tagged proteins, fluorescence resonance energy transfer microscopy showed a stable physical interaction between the G-protein alpha subunit and RGS (RGS8 and RGS7) that is independent of the functional state of the G-protein. RGS8 does not directly interact with G-protein-coupled receptors. Our data show participation of the RGS in the ternary complex between agonist-receptor and G-protein to form a "quaternary complex." Thus we propose a novel model for the action of RGS proteins in the G-protein cycle in which the RGS protein appears to enhance the "kinetic efficacy" of the ternary complex, by direct association with the G-protein alpha subunit.
Sarcomeric gene mutations frequently underlie hypertrophic cardiomyopathy (HCM), a prevalent and complex condition leading to left ventricle thickening and heart dysfunction. We evaluated isogenic genome-edited human pluripotent stem cell-cardiomyocytes (hPSC-CM) for their validity to model, and add clarity to, HCM. CRISPR/Cas9 editing produced 11 variants of the HCM-causing mutation c.C9123T-MYH7 [(p.R453C-β-myosin heavy chain (MHC)] in 3 independent hPSC lines. Isogenic sets were differentiated to hPSC-CMs for high-throughput, non-subjective molecular and functional assessment using 12 approaches in 2D monolayers and/or 3D engineered heart tissues. Although immature, edited hPSC-CMs exhibited the main hallmarks of HCM (hypertrophy, multi-nucleation, hypertrophic marker expression, sarcomeric disarray). Functional evaluation supported the energy depletion model due to higher metabolic respiration activity, accompanied by abnormalities in calcium handling, arrhythmias, and contraction force. Partial phenotypic rescue was achieved with ranolazine but not omecamtiv mecarbil, while RNAseq highlighted potentially novel molecular targets. Our holistic and comprehensive approach showed that energy depletion affected core cardiomyocyte functionality. The engineered R453C-βMHC-mutation triggered compensatory responses in hPSC-CMs, causing increased ATP production and αMHC to energy-efficient βMHC switching. We showed that pharmacological rescue of arrhythmias was possible, while MHY7: MYH6 and mutant: wild-type MYH7 ratios may be diagnostic, and previously undescribed lncRNAs and gene modifiers are suggestive of new mechanisms.
LQTS (long QT syndrome) is an important cause of cardiac sudden death. LQTS is characterized by a prolongation of the QT interval on an electrocardiogram. This prolongation predisposes the individual to torsade-de-pointes and subsequent sudden death by ventricular fibrillation. Mutations in a number of genes that encode ion channels have been implicated in LQTS. Hereditary mutations in the alpha- and beta-subunits, KCNQ1 and KCNE1 respectively, of the K(+) channel pore I(Ks) are the commonest cause of LQTS and account for LQTS types 1 and 5 respectively (LQT1 and LQT5). Recently, it has been shown that disease pathogenesis in LQT1 can be influenced by the abnormal trafficking of KCNQ1. In comparison, whether defective trafficking of KCNE1 plays a role in LQT5 is less well established.
The activation of ATP-sensitive K+ channels by protein kinase A in vascular smooth muscle is an important component of the action of vasodilators. In this study, we examine the molecular mechanisms of regulation of the cloned equivalent of this channel comprising the sulfonylurea receptor 2B and the inward rectifier 6.1 subunit (SUR2B/Kir6.1). Specifically, we focus on whether the channel is directly phosphorylated and the sites at which this occurs in the protein complex. We identify one site in Kir6.1 (S385) and two sites in SUR2B (T633 and S1465) using a combination of biochemical and functional assays. Our work supports a model in which multiple sites in the channel complex have to be phosphorylated before activation occurs.
Abstract Styrene has been polymerized thermally at 60.5°C, without added initiator, in the presence of ferrocene. An increase in the rate of polymerization was observed, but the degree of polymerization remained constant. Evidence of ferrocene–styrene interaction is presented, and a kinetic scheme involving propagation with styrene in such an interaction state is proposed to account for these findings. Various kinetic parameters defined by this scheme have been evaluated.
Background and Aim: Two mRNA isoforms of BNP (i.e., immature and mature transcripts) were found to be expressed in both normal and diseased porcine heart. Given that there were indications suggesting that heart failure (HF) may be associated with production of various isoforms of BNP, we searched for novel variants of alternative splicing of BNP in the porcine model of HF. Methods: HF was induced in 6-day-old neonatal piglets by a single i.v. injection of 2 mg/kg of Doxorubicin (Dox). To profile the cardiac gene expression changes associated with Dox-induced HF, a non-radioactive mRNA differential display (DDRT-PCR) was used. A novel alternative spliced variant of the BNP gene (designated E2-BNP) was identified, characterized, cloned and expressed. E2-BNP mRNA levels were determined by semi-quantitative and qRT-PCR. Results: On day 24 after Dox-administration, experimented piglets developed the features of diastolic HF. Using DDRT-PCR, the band corresponding to the E2-BNP was identified as being overexpressed in failing versus normal piglet myocardium. The sequence of this band displayed a 100% homology with exon 1 and exon 3 sequences of BNP. Further RT-PCR amplification of BNP (through exons 1-3) from the failing piglet myocardium cDNA produced three products, two of which were identical to the reported BNP immature and mature transcripts, whereas the third one was characterized by the precise deletion of the exon 2. Skipping of exon 2 ( E2) causes a frame-shift from the beginning of exon 3, generating a coding sequence for a new protein with no C-terminal homology to known natriuretic peptides. In newborn piglets (n=5), the E2-BNP mRNA was 15 fold more abundant in the left ventricle (LV) than in the right ventricle (RV). During development, the E2-BNP gene expression is rapidly downregulated in both ventricles being, at postnatal day 30, 10-20-fold lower as compared to newborn piglets. By contrast, in 30-day-old piglets with Dox-induced HF (n=12) the E2-BNP mRNA levels were found to be consistently and significantly augmented in both ventricles being much more up-regulated in the RV (40±2.6 fold) than in the LV (10±1.6 fold) as compared with age-matched controls. The average fold-increase of the BNP mRNA content in the failing porcine RV and LV was only 6.3±0.5 and 5.1±0.3, respectively. Conclusion: As such, variation in E2-BNP mRNA transcript abundance within the ventricular wall can be used to diagnose heart conditions in large-animal models of HF. In addition, the E2-BNP seems to be superior to BNP as a molecular marker for HF.
Abstract The electrocardiographic PR interval reflects atrioventricular conduction, and is associated with conduction abnormalities, pacemaker implantation, atrial fibrillation (AF), and cardiovascular mortality 1,2 . We performed multi-ancestry (N=293,051) and European only (N=271,570) genome-wide association (GWAS) meta-analyses for the PR interval, discovering 210 loci of which 149 are novel. Variants at all loci nearly doubled the percentage of heritability explained, from 33.5% to 62.6%. We observed enrichment for genes involved in cardiac muscle development/contraction and the cytoskeleton highlighting key regulation processes for atrioventricular conduction. Additionally, 19 novel loci harbour genes underlying inherited monogenic heart diseases suggesting the role of these genes in cardiovascular pathology in the general population. We showed that polygenic predisposition to PR interval duration is an endophenotype for cardiovascular disease risk, including distal conduction disease, AF, atrioventricular pre-excitation, non-ischemic cardiomyopathy, and coronary heart disease. These findings advance our understanding of the polygenic basis of cardiac conduction, and the genetic relationship between PR interval duration and cardiovascular disease.
Abstract Stillbirth is the loss of a fetus after 22 weeks of gestation, of which almost half go completely unexplained despite post-mortem. We recently sequenced 35 arrhythmia-associated genes from 70 unexplained stillbirth cases. Our hypothesis was that deleterious mutations in channelopathy genes may have a functional effect in utero that may be pro-arrhythmic in the developing fetus. We observed four heterozygous, nonsynonymous variants in transient receptor potential melastatin 7 (TRPM7), a ubiquitously expressed ion channel known to regulate cardiac development and repolarization in mice. We used site-directed mutagenesis and single-cell patch-clamp to analyze the functional effect of the four stillbirth mutants on TRPM7 ion channel function in heterologous cells. We also used cardiomyocytes derived from human pluripotent stem cells to model the contribution of TRPM7 to action potential morphology. Our results show that two TRPM7 variants, p.G179V and p.T860M, lead to a marked reduction in ion channel conductance. This observation was underpinned by a lack of measurable TRPM7 protein expression, which in the case of p.T860M was due to rapid proteasomal degradation. We also report that human hiPSC-derived cardiomyocytes possess measurable TRPM7 currents; however, siRNA knockdown did not directly affect action potential morphology. TRPM7 variants found in the unexplained stillbirth population adversely affect ion channel function and this may precipitate fatal arrhythmia in utero.
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