The effects of the Rgs6 on HRV are mediated by the I_KACh and are influenced by the m₂R activity.

<p>A, Schematic representation of the pathway targeted both genetically and pharmacologically. Abbreviations are: atropine (Atro), carbamylcholine (CCh). B, Effect of m₂R blockade by atropine on HRV in wild-type (black; n = 7) and <i>Rgs6^−/−</i> hearts (red; n = 10). No significant effect of drug was observed in wild-type hearts. C, Increased sensitivity of <i>Rgs6^−/−</i> hearts to m₂R stimulation and its rescue by I_KACh (<i>Girk4</i>) ablation. Increasing concentrations of CCh were applied to isolated perfused hearts (n = 4–6 per genotype). D, m₂R stimulation non-proportionately increased HRV in <i>Rgs6^−/−</i> hearts. Hearts (n = 3–6 per genotype) were perfused with CCh (∼IC₁₀ concentration) identified from dose-response studies, followed by measurement of changes in the RMSSD parameters. Symbols: * P<0.05 vs wild-type, #P<0.05 vs treatment.</p

In Situ Maleimide Bridging of Disulfides and a New Approach to Protein PEGylation

The introduction of non-natural entities into proteins by chemical modification has numerous applications in fundamental biological science and for the development and manipulation of peptide and protein therapeutics. The reduction of native disulfide bonds provides a convenient method to access two nucleophilic cysteine residues that can serve as ideal attachment points for such chemical modification. The optimum bioconjugation strategy utilizing these cysteine residues should include the reconstruction of a bridge to mimic the role of the disulfide bond, maintaining structure and stability of the protein. Furthermore, the bridging chemical modification should be as rapid as possible to prevent problems associated with protein unfolding, aggregation, or disulfide scrambling. This study reports on an in situ disulfide reduction-bridging strategy that ensures rapid sequestration of the free cysteine residues in a bridge, using dithiomaleimides. This approach is then used to PEGylate the peptide hormone somatostatin and retention of biological activity is demonstrated

Rgs6 and Girk4 have opposite effects on HRV in isolated hearts.

<p>A, Average HR in hearts isolated from wild-type (wt, n = 36), <i>Rgs6</i>^−/− (n = 52), and <i>Girk4</i>^−/− (n = 19) mice. B, ECG traces recorded in isolated wild-type (black), <i>Rgs6^−/−</i> (red), and <i>Girk4^−/−</i> (green) hearts. Note rhythm irregularity in <i>Rgs6</i>^−/− hearts. C, Quantification of sinoatrial dysfunction events. D–F, Representative tachograms of baseline ECG in wild-type (black), <i>Rgs6</i>^−/− (red), and <i>Girk4</i>^−/− (green) hearts. G–I, Key HRV parameters in the time and frequency domains from ECG recordings. J–L, Non-linear HRV analysis by Poincare plots for wild-type (J), <i>Rgs6^−/−</i> (K), and <i>Girk4^−/−</i> (L) hearts. Symbols: * P<0.05, ** P<0.01, ***P<0.001 vs. wild-type.</p

Essential Role of the m₂R-RGS6-I_KACh Pathway in Controlling Intrinsic Heart Rate Variability

<div><p>Normal heart function requires generation of a regular rhythm by sinoatrial pacemaker cells and the alteration of this spontaneous heart rate by the autonomic input to match physiological demand. However, the molecular mechanisms that ensure consistent periodicity of cardiac contractions and fine tuning of this process by autonomic system are not completely understood.</p><p>Here we examined the contribution of the m₂R-I_KACh intracellular signaling pathway, which mediates the negative chronotropic effect of parasympathetic stimulation, to the regulation of the cardiac pacemaking rhythm. Using isolated heart preparations and single-cell recordings we show that the m₂R-I_KACh signaling pathway controls the excitability and firing pattern of the sinoatrial cardiomyocytes and determines variability of cardiac rhythm in a manner independent from the autonomic input. Ablation of the major regulator of this pathway, Rgs6, in mice results in irregular cardiac rhythmicity and increases susceptibility to atrial fibrillation. We further identify several human subjects with variants in the <i>RGS6</i> gene and show that the loss of function in RGS6 correlates with increased heart rate variability. These findings identify the essential role of the m₂R-I_KACh signaling pathway in the regulation of cardiac sinus rhythm and implicate RGS6 in arrhythmia pathogenesis.</p></div

Abnormal sinus arrhythmia in a human subject with dysfunctional RGS6.

<p>A, HRV measured in humans carrying variants in <i>RGS6</i> and 11 age-matched control subjects (wt, black). Lines represent upper (2σ) and lower (−2 σ) 95% confidence thresholds as determined by the 2σ rule. <i>Insert</i>: domain structure of RGS6 protein. Arrows show localization of corresponding variants. B, Representative tachograms of RR intervals from a control subject (black) and a subject heterozygous for the p.Val13LeufsX11 variant in the <i>RGS6</i> gene (red) determined from continuous Holter recordings. C, Schematics of the assay design to study effects of mutations on the RGS6 function. Stimulation of the m₂R by ACh results in the dissociation of Gμo from the heterotrimer. Released Gβγ subunits tagged with Venus become available for the interaction with Nluc8-tagged GRK reporter producing the BRET signal. D. Representative responses to sequential application of ACh (10 µM) and atropine (1 mM) recorded in the presence of the indicated constructs. The BRET signals averaged from 4 experiments were plotted as individual data points. <i>E</i>, Catalytic activity of RGS6 defined by the <i>k</i>_GAP parameter. To determine the <i>k</i>_GAP values, the deactivation rate constant measured in the absence of RGS6 was subtracted from values measured in the presence of RGS6. Symbols: ***, p<0.001 (n = 4).</p

Inactivation of <i>Rgs6</i> disrupts cardiac rhythm in mice.

<p>A, Representative tachograms of RR intervals from wild-type (black) and <i>Rgs6^−/−</i> (red) mice recorded by ECG radiotelemetry. B and C, Summary of HRV analysis in conscious, freely-moving mice. D, Burst pacing induced AF in <i>Rgs6^−/−</i> but not in wild-type mice. Note an irregular rhythm with no discernible P waves in the <i>Rgs6^−/−</i> recording. E, Quantification of AF induction probability. Symbols: *, P<0.05.</p

Ablation of <i>Rgs6</i> reduces excitability of sinoatrial cells and disrupts their automaticity.

<p>A, Resting membrane potential measured immediately after obtaining whole-cell access in wild-type (wt), <i>Rgs6^−/−</i>, and <i>Girk4^−/−</i> SAN cells. B, Inward currents evoked by application of acetylcholine (ACh, 100 µM) in SAN cells from wild-type (black), <i>Rgs6^−/−</i> (red) and <i>Girk4^−/−</i> (green, no current) mice. C, Summary of steady-state ACh-induced deactivation kinetics of I_KACh in wild-type and <i>Rgs6^−/−</i> SAN cells (n = 11–15 cells/genotype). D, Representative traces of spontaneous calcium oscillations recorded from wild-type (black; n = 14) and <i>Rgs6^−/−</i> (red, n = 20) SAN cells. Arrows show skipped beats. E, Quantification of SAN arrhythmic events defined as more than 15% change in duration of peak-to-peak interval of spontaneous calcium oscillations in wild-type (n = 11) and <i>Rgs6^−/−</i> (n = 17) SAN cells. F, Reduced frequency of spontaneous calcium oscillations recorded in <i>Rgs6^−/−</i> SAN cardiomyocytes as compared to wild-type (n = 14–20 cells/genotype). G, Increased variability of spontaneous calcium oscillations in <i>Rgs6^−/−</i> SAN cells as determined by increase in RMSSD values (n = 14–20 cells per genotype). Symbols: *P<0.05; **P<0.01; ***P<0.001.</p

DataSheet1_Omega-3 polyunsaturated fatty acid-induced vasodilation in mouse aorta and mesenteric arteries is not mediated by ATP-sensitive potassium channels.PDF

There is strong evidence that the omega-3 polyunsaturated fatty acids (n-3 PUFAs) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) have cardioprotective effects. n-3 PUFAs cause vasodilation in hypertensive patients, in part controlled by increased membrane conductance to potassium. As KATP channels play a major role in vascular tone regulation and are involved in hypertension, we aimed to verify whether n-3 PUFA-mediated vasodilation involved the opening of KATP channels. We used a murine model in which the KATP channel pore subunit, Kir6.1, is deleted in vascular smooth muscle. The vasomotor response of preconstricted arteries to physiologically relevant concentrations of DHA and EPA was measured using wire myography, using the channel blocker PNU-37883A. The effect of n-3 PUFAs on potassium currents in wild-type native smooth muscle cells was investigated using whole-cell patch clamping. DHA and EPA induced vasodilation in mouse aorta and mesenteric arteries; relaxations in the aorta were sensitive to KATP blockade with PNU-37883A. Endothelium removal didn’t affect relaxation to EPA and caused a small but significant inhibition of relaxation to DHA. In the knock-out model, relaxations to DHA and EPA were unaffected by channel knockdown but were still inhibited by PNU-37883A, indicating that the action of PNU-37883A on relaxation may not reflect inhibition of KATP. In native aortic smooth muscle cells DHA failed to activate KATP currents. We conclude that DHA and EPA cause vasodilation in mouse aorta and mesenteric arteries. Relaxations in blocker-treated arteries from knock-out mice demonstrate that KATP channels are not involved in the n-3 PUFA-induced relaxation.</p

Data_Sheet_1_Investigating the Complex Arrhythmic Phenotype Caused by the Gain-of-Function Mutation KCNQ1-G229D.pdf

The congenital long QT syndrome (LQTS) is a cardiac electrophysiological disorder that can cause sudden cardiac death. LQT1 is a subtype of LQTS caused by mutations in KCNQ1, affecting the slow delayed-rectifier potassium current (IKs), which is essential for cardiac repolarization. Paradoxically, gain-of-function mutations in KCNQ1 have been reported to cause borderline QT prolongation, atrial fibrillation (AF), sinus bradycardia, and sudden death, however, the mechanisms are not well understood. The goal of the study is to investigate the ionic, cellular and tissue mechanisms underlying the complex phenotype of a gain-of-function mutation in KCNQ1, c.686G > A (p.G229D) using computer modeling and simulations informed by in vitro measurements. Previous studies have shown this mutation to cause AF and borderline QT prolongation. We report a clinical description of a family that carry this mutation and that a member of the family died suddenly during sleep at 21 years old. Using patch-clamp experiments, we confirm that KCNQ1-G229D causes a significant gain in channel function. We introduce the effect of the mutation in populations of atrial, ventricular and sinus node (SN) cell models to investigate mechanisms underlying phenotypic variability. In a population of human atrial and ventricular cell models and tissue, the presence of KCNQ1-G229D predominantly shortens atrial action potential duration (APD). However, in a subset of models, KCNQ1-G229D can act to prolong ventricular APD by up to 7% (19 ms) and underlie depolarization abnormalities, which could promote QT prolongation and conduction delays. Interestingly, APD prolongations were predominantly seen at slow pacing cycle lengths (CL > 1,000 ms), which suggests a greater arrhythmic risk during bradycardia, and is consistent with the observed sudden death during sleep. In a population of human SN cell models, the KCNQ1-G229D mutation results in slow/abnormal sinus rhythm, and we identify that a stronger L-type calcium current enables the SN to be more robust to the mutation. In conclusion, our computational modeling experiments provide novel mechanistic explanations for the observed borderline QT prolongation, and predict that KCNQ1-G229D could underlie SN dysfunction and conduction delays. The mechanisms revealed in the study can potentially inform management and treatment of KCNQ1 gain-of-function mutation carriers.</p

Discovery of novel heart rate-associated loci using the Exome Chip

Resting heart rate is a heritable trait, and an increase in heart rate is associated with increased mortality risk. Genome-wide association study analyses have found loci associated with resting heart rate, at the time of our study these loci explained 0.9% of the variation. This study aims to discover new genetic loci associated with heart rate from Exome Chip meta-analyses.Heart rate was measured from either elecrtrocardiograms or pulse recordings. We meta-analysed heart rate association results from 104 452 European-ancestry individuals from 30 cohorts, genotyped using the Exome Chip. Twenty-four variants were selected for follow-up in an independent dataset (UK Biobank, N = 134 251). Conditional and gene-based testing was undertaken, and variants were investigated with bioinformatics methods.We discovered five novel heart rate loci, and one new independent low-frequency non-synonymous variant in an established heart rate locus (KIAA1755). Lead variants in four of the novel loci are non-synonymous variants in the genes C10orf71, DALDR3, TESK2 and SEC31B. The variant at SEC31B is significantly associated with SEC31B expression in heart and tibial nerve tissue. Further candidate genes were detected from long-range regulatory chromatin interactions in heart tissue (SCD, SLF2 and MAPK8). We observed significant enrichment in DNase I hypersensitive sites in fetal heart and lung. Moreover, enrichment was seen for the first time in human neuronal progenitor cells (derived from embryonic stem cells) and fetal muscle samples by including our novel variants.Our findings advance the knowledge of the genetic architecture of heart rate, and indicate new candidate genes for follow-up functional studies