AIMS:Loss-of-function (LOF) mutations of the cardiac Na+ channel (SCN5A) are causatively associated with the Brugada Syndrome (BrS). However, the onset of Ventricular Fibrillation (VF) is a rare event, and critical factors favouring the pathological phenotype remain often elusive. This study explores how concomitant triggering conditions may impact on VF onset in a symptomatic proband carrying the S805L/SCN5A BrS mutation. METHODS AND RESULTS:Clinical, in-vitro, numerical, and structural analyses were performed. A 67-year-old male was resuscitated after cardiac arrest, and clinical analysis upon hospitalisation revealed severe hypokalaemia (2.5 mEq/L). The ECG showed a coved type-I BrS pattern and the SCN5A mutation (S805L) was identified. Patch-clamp studies carried out in a heterologous expression system (HEK293 cells) revealed that WT/S805L channels exhibit two different phenotypes (normal and LOF); the main parameter controlling this distribution is the cell membrane potential. A protected/normal behaviour was observed at -80 mV; conversely, LOF occurred at more negative potentials (-100/-120 mV). Further analyses in isolated outflow tract ventricular cardiomyocytes showed that hypokalaemia (and bradycardia) induced diastolic potential hyperpolarisation, thus favouring the Na+ current LOF. Computational and molecular modelling confirmed our findings and revealed the structural determinant of this alteration. CONCLUSION:WT/S805L Na+ channels exhibit either a LOF or a wild-type-like behaviour depending on the membrane potential. Since hypokalaemia and slow pacing rate induce cell hyperpolarisation and the associated LOF, they represent concurrent elements creating the scenario responsible for the VF and cardiac arrest. These results may represent an interpretative paradigm applicable to other BrS mutations.

AIM:This study aims to investigate whether the Fibroblast Growth Factor 23 (FGF23) modulates the electrical activity of sinoatrial (SAN) cells. The canonical function of FGF23 is to regulate body phosphorus and calcium homeostasis by activating the FGF1 receptors (FGFR1)/α-Klotho complex in the kidney and parathyroid glands. High levels of FGF23 can induce cardiac arrhythmias by affecting cardiomyocyte's function in an α-Klotho independent manner. Although SAN cells are not traditionally considered targets of FGF23, the presence of α-Klotho in pacemaker and not in ventricular cells has raised this possibility. METHODS:The effect of FGF23 was evaluated by patch-clamp experiments on mouse SAN and on human-induced pluripotent stem cells-derived pacemaker-like cardiomyocytes (hiPSC-derived pCMs). RESULTS:Our data reveal that mouse SAN cells express both membrane α-Klotho and FGF23 receptors (FGFR) and that 48 h tissue incubation with FGF23 (10 ng/mL) increases the spontaneous action potential (AP) frequency of these cells through an increase in the funny If current. Patch-clamp experiments carried out using the pan-FGFR inhibitor, PD173074, and SAN cells isolated from α-Klotho hypomorphic mice suggested that FGF23 effects are mediated by the activation of the FGFR-α-Klotho complex. FGFRs expression data and FGF23-induced electrical modification were further confirmed in hiPSC-derived pCMs. Indeed, 48 h incubation of these cells with FGF23 increases both the AP frequency, in a dose-dependent manner, and the If current. CONCLUSIONS:This study represents the first evidence that FGF23 directly regulates the SAN electrical activity.

Camizestrant, a next-generation selective estrogen receptor (ER) degrader and complete ER antagonist, has been associated with a reversible dose- and time-dependent heart rate (HR) reduction in clinical studies. This nonclinical investigation aimed to understand the mechanism of camizestrant-induced HR reduction. The effects of camizestrant on HR in vivo were assessed in rat and dog telemetry studies. Effects on pacemaker channel function in vitro were assessed using patch-clamp electrophysiology in Chinese hamster ovary cells expressing human hyperpolarization-activated cyclic nucleotide-gated channel 4 (hHCN4), human embryonic stem cell (hESC)-derived sinoatrial node (SAN) cardiomyocytes, and primary rat SAN cardiomyocytes. In dogs, 28-day repeat-dose camizestrant administration caused a reversible dose- and time-dependent HR reduction (maximum reduction of 53 beats per min [bpm] on Day 25 vs pre-study levels at 20 mg/kg). HR reduction was also noted in rats (maximum reduction 89 bpm vs vehicle [23%] on Day 5 of a 7-day study at 75 mg/kg). Responses to chronotropic stimuli (e.g., atropine and isoprenaline) were reduced in dogs treated with camizestrant. Camizestrant-induced HR reduction was still present following combined sympathetic (atenolol) and parasympathetic (atropine) inhibition in dogs, as well as vagotomy in rats. Camizestrant reduced hHCN4 current density in Chinese hamster ovary cells, as well as beat rate and "funny" pacemaker (If) current activity in hESC-derived SAN cardiomyocytes. Camizestrant at 75 mg/kg for 7 days significantly reduced If current activity versus vehicle in isolated SAN cardiomyocytes. These results support the hypothesis that camizestrant exerts a pharmacologic, reversible reduction in HR by decreasing SAN pacemaker current activity.

Given that the immunity imbalance in patients with Long COVID-19 (LC) may pose a significant global health and economic post-pandemic burden, there is an emergent need to identify therapeutic targets and treatment options. Traditional Chinese medicine (TCM), as an evidence-based therapeutic approach, can effectively improve the sequelae of LC patients by eliminating pathogenic factors. The purpose of this paper is to discuss how immune remodeling contributes to the pathogenesis of LC, the clinical evidence supporting TCM’s treatment of LC, and the mechanism of TCM modulating immune remodeling and relieving chronic inflammation to develop new ideas for the treatment of LC as well as the development of drugs. Data were retrieved using appropriate keywords from a variety of internet databases, including PubMed and Web of Science. Current evidence shows that LC can affect multiple organ systems, and its prominent manifestations include respiratory complications, neurological symptoms and cardiovascular dysfunction. Immunoassay showed a characteristic increase in interleukin 6 (IL-6), interferon gamma (IFN-γ), and T helper (Th)17/regulatory T (Treg) imbalance. TCM interventions have shown great therapeutic potential, with active compounds such as baicalin reducing lung inflammation and ginsenosides improving heart function. Clinical research reports that Qingfei Paidu decoction (QFPD) can effectively alleviate respiratory symptoms, and Sini powder (SNP) has antidepressant effects. TCM interventions can be tailored based on the specific clinical symptoms of individual patients. This article elucidates the crucial role of inflammation and immune dysfunction in alleviating multiple organ symptoms of LC. TCM used in LC treatment is an important source of new molecules. These new molecules may act synergistically to combat adverse effects such as COVID-19 infection-induced inflammation and oxidative stress.

Brugada Syndrome (BrS) is a genetic disease associated with ventricular arrhythmias and is one of the causes of sudden cardiac death. In particular, dysfunctional cardiac Na + channels (SCN5A) represent the only mechanism supporting the autosomal dominant inheritance. A proband without previous medical history was referred to the intensive care unit after resuscitation showing ECG Type 1 BrS pattern and a significant hypokalemia (2.5 mEq/L). Genetic analysis identified a SCN5A de-novo mutation (S805L) in heterozygosis. This study investigates the causative association between the S805L mutation and the BrS event. First, we expressed wild type (WT) and/or mutated (Hetero and Homo) channels in HEK293T. The application of I/V protocols (hp -120 mV) revealed reduced I Na density in Homo and Hetero (-65% and -26% vs WT at -20 mV), indicating S805L as a loss-of-function (LOF) mutation. However, Hetero I Na density was comparable to WT at more depolarized hp (-80 mV), suggesting alterations in the voltage dependent availability of the channel. Accordingly, steady-state inactivation curves of both Hetero and Homo I Na were right shifted, indicating a gain-of-function (GOF) behaviour (Hetero availability: +13% vs WT at -80 mV). To investigate the possible role of hypokalemia in uncovering the LOF behaviour of the mutation, we reproduced in vitro the hypokalemic condition of the patient testing the effects of external K + (K + out ) on diastolic membrane potential (E diast ) of paced guinea-pig cardiomyocytes. Cells were isolated from the Right Ventricular Outflow Tract, known as site of BrS onset. In comparison to a normokalemic condition, E diast significantly hyperpolarized at 2.5 mM K + out and the effect was greater at low pacing rates (1 Hz) (-16.5 mV vs E diast at 5 mM K + and 4 Hz). Thus, the combination of bradycardia and hypokalemia might represent a mechanism able to unmask the LOF effect of S805L mutation in Hetero conditions. Computational approach on the human ventricular action potential confirmed these findings. In conclusion, S805L-SCN5A is, at the same time, a LOF and GOF mutation causing reduced channel expression with increased channel availability. While the balance between GOF-LOF effects guaranties a “protected” phenotype, the hypokalemic-induced cell hyperpolarization might reduce the GOF, leading to the appearance of the BrS event.

Tongmai Yangxin (TMYX) is a complex compound of the Traditional Chinese Medicine (TCM) used to treat several cardiac rhythm disorders; however, no information regarding its mechanism of action is available. In this study we provide a detailed characterization of the effects of TMYX on the electrical activity of pacemaker cells and unravel its mechanism of action. Single-cell electrophysiology revealed that TMYX elicits a reversible and dose-dependent (2/6 mg/ml) slowing of spontaneous action potentials rate (−20.8/–50.2%) by a selective reduction of the diastolic phase (−50.1/–76.0%). This action is mediated by a negative shift of the If activation curve (−6.7/–11.9 mV) and is caused by a reduction of the cyclic adenosine monophosphate (cAMP)-induced stimulation of pacemaker channels. We provide evidence that TMYX acts by directly antagonizing the cAMP-induced allosteric modulation of the pacemaker channels. Noticeably, this mechanism functionally resembles the pharmacological actions of muscarinic stimulation or β-blockers, but it does not require generalized changes in cytoplasmic cAMP levels thus ensuring a selective action on rate. In agreement with a competitive inhibition mechanism, TMYX exerts its maximal antagonistic action at submaximal cAMP concentrations and then progressively becomes less effective thus ensuring a full contribution of If to pacemaker rate during high metabolic demand and sympathetic stimulation.

Tongmai Yangxin (TMYX), is a complex compound of a Traditional Chinese Medicine (TCM) used to treat several cardiac rhythm disorders; however, no information regarding its mechanism of action is available. In this study we provide a detailed characterization of the effects of TMYX on the electrical activity of pacemaker cells and unravel its mechanism of action. Single-cell electrophysiology revealed that TMYX elicits a reversible and dose-dependent (2/6 mg/ml) slowing of spontaneous action potentials rate (−20.8/-50.2%) by a selective reduction of the diastolic phase (−50.1/-76.0%). This action is mediated by a negative shift of the I f activation curve (−6.7/-11.9 mV) and is caused by a reduction of the cAMP-induced stimulation of pacemaker channels. We provide evidence that TMYX acts by directly antagonizes the cAMP-induced allosteric modulation of the pacemaker channels. Noticeably, this mechanism functionally resembles the pharmacological actions of muscarinic stimulation or β-blockers, but it does not require generalized changes in cytoplasmic cAMP levels thus ensuring a selective action on rate. In agreement with a competitive inhibition mechanism, TMYX exerts its maximal antagonistic action at submaximal cAMP concentrations and then progressively becomes less effective thus ensuring a full contribution of I f to pacemaker rate during high metabolic demand and sympathetic stimulation. Funding sources This work was supported by grants from Tianjin Zhongxin Pharmaceutical Group Co., Ltd. Le Ren Tang Pharmaceutical Factory P.R. China. The financial supporter played no role in the study design, data collection and analysis, decision to publish, or the preparation of the manuscript.

Objective The aim of this study was to assess age-related changes in cardiac autonomic modulation and heart rate variability (HRV) and their association with spontaneous and pharmacologically induced vulnerability to cardiac arrhythmias, to verify the translational relevance of mouse models for further in-depth evaluation of the link between autonomic changes and increased arrhythmic risk with advancing age. Methods Heart rate (HR) and time- and frequency-domain indexes of HRV were calculated from Electrocardiogram (ECG) recordings in two groups of conscious mice of different ages (4 and 19 months old) (i) during daily undisturbed conditions, (ii) following peripheral β-adrenergic (atenolol), muscarinic (methylscopolamine), and β-adrenergic + muscarinic blockades, and (iii) following β-adrenergic (isoprenaline) stimulation. Vulnerability to arrhythmias was evaluated during daily undisturbed conditions and following β-adrenergic stimulation. Results HRV analysis and HR responses to autonomic blockades revealed that 19-month-old mice had a lower vagal modulation of cardiac function compared with 4-month-old mice. This age-related autonomic effect was not reflected in changes in HR, since intrinsic HR was lower in 19-month-old compared with 4-month-old mice. Both time- and frequency-domain HRV indexes were reduced following muscarinic, but not β-adrenergic blockade in younger mice, and to a lesser extent in older mice, suggesting that HRV is largely modulated by vagal tone in mice. Finally, 19-month-old mice showed a larger vulnerability to both spontaneous and isoprenaline-induced arrhythmias. Conclusion The present study combines HRV analysis and selective pharmacological autonomic blockades to document an age-related impairment in cardiac vagal modulation in mice which is consistent with the human condition. Given their short life span, mice could be further exploited as an aged model for studying the trajectory of vagal decline with advancing age using HRV measures, and the mechanisms underlying its association with proarrhythmic remodeling of the senescent heart.

Background: Brugada syndrome (BrS) is a cardiac disorder characterized by conduction abnormalities that can lead to sudden death; syncope and cardiac arrest are clinical manifestations which are often associated with an enhancement of the vagal activity. Mutations in the SCN5A gene (Na v 1.5 channel) are the most common cause of the inherited forms of BrS. Objective: To characterize the functional behavior of mutant Na v 1.5 channels expressing a novel heterozygous mutation (S805L) recently identified in an Italian family affected by the BrS. Methods: HEK cells were used as experimental model to express both the wild-type (WT) and the mutated S805L channels (alone, Homo or in combination, Hetero) and the accessory β-subunit (SCN1B). Patch-clamp and western blot experiments were carried out to assess the dysfunctional role of the mutation. Results: When compared to the WT current, the S508L mutation significantly (P&It0.05) decreases the peak current density by about 65% for the Homo condition (WT: -120.2±10.2, n=28); Homo: -40.3±4.2, n=16) and by 35% for the Hetero condition (Hetero: -78.2±8.3, n=27). Densitometric analysis carried out on western blot data further support the conclusion that S805L channels are less abundant in the plasma membrane. We also observed that the S805L mutation positively shifts the V½ values of the voltage dependence of the inactivation of both Homo and Hetero currents (V½: WT -85.5±0.2 mV, n=55; Homo -80.9±0.3 mV, n=22; Hetero -81.9±0.2 mV, n=25; P&It0.05); a positive shift of the V½ of the activation was also observed but only in the Homo condition (V½: WT -33.0±0.4 mV, n=28; Homo -30.0±0.5, n=16, P&It0.05). The kinetics of recovery from inactivation and the amplitude of the late sodium current were also evaluated but they were unaffected by the mutation. Conclusion: When expressed in the Hetero condition, the S805L mutation causes a reduction in the channel expression, however, the positive shift of the inactivation curve suggests an increase in Na channel availability. We thus believe that the precise quantitative balance between these two phenomena and their relation with vagal activity may underlie the clinical manifestation of the disease.