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.

Doxorubicin (Doxo) is an anthracycline widely used as a chemotherapeutic agent for many solid and hematological cancers. Its clinical use is limited due to a cumulative dose-dependent and irreversible cardiotoxicity that can cause progressive cardiomyopathy and congestive heart failure. A cardioprotective therapy that can decrease heart damage without reducing the anticancer efficacy during Doxo therapy is of utmost importance. Anthocyanins (ACNs) are renowned cardioprotective agents thanks to their antioxidant and anti-inflammatory properties. An ACN-rich diet from purple corn, which mainly contains cyanidin 3-glucoside (C3G) and its acetylated derivatives, has been previously shown to be effective in reducing Doxo-induced cardiotoxicity in mice. Aiming at unveiling the molecular mechanisms involved in ACN protection, we considered the fibroblast growth factor 21/AMP-activated protein kinase/SIRTUIN1 (FGF21/AMPK/SIRT1)/p53 pathway in murine HL-1 cardiomyocytes treated with Doxo in the presence or absence of purple corn extract (RED). Our work shows that Doxo-induced AMPK activation is restored to control levels by the RED extract. p53 acetylation was increased by the RED extract and upon Sirt1 silencing, indicating that p53 acetylation is SIRT1-dependent and suggesting that the RED extract may affect SIRT1 activity through AMPK. Notably, increased p53 acetylation led to decreased levels of cleaved-caspase 3 and Puma and p21 transcript levels, indicating a reduced level of apoptosis. The RED-induced cardioprotection and p53 acetylation were confirmed in mouse primary cardiomyocytes. In conclusion, the RED extract may prevent cardiomyocytes apoptosis through the modulation of AMPK and acetylation of p53.

Caveolae constitute membrane microdomains where receptors and ion channels functionally interact. Caveolin-3 (cav-3) is the key structural component of muscular caveolae. Mutations in CAV3 lead to caveolinopathies, which result in both muscular dystrophies and cardiac diseases. In cardiomyocytes, cav-1 participates with cav-3 to form caveolae; skeletal myotubes and adult skeletal fibers do not express cav-1. In the heart, the absence of cardiac alterations in the majority of cases may depend on a conserved organization of caveolae thanks to the expression of cav-1. We decided to focus on three specific cav-3 mutations (Δ62-64YTT; T78K and W101C) found in heterozygosis in patients suffering from skeletal muscle disorders. We overexpressed both the WT and mutated cav-3 together with ion channels interacting with and modulated by cav-3. Patch-clamp analysis conducted in caveolin-free cells (MEF-KO), revealed that the T78K mutant is dominant negative, causing its intracellular retention together with cav-3 WT, and inducing a significant reduction in current densities of all three ion channels tested. The other cav-3 mutations did not cause significant alterations. Mathematical modelling of the effects of cav-3 T78K would impair repolarization to levels incompatible with life. For this reason, we decided to compare the effects of this mutation in other cell lines that endogenously express cav-1 (MEF-STO and CHO cells) and to modulate cav-1 expression with an shRNA approach. In these systems, the membrane localization of cav-3 T78K was rescued in the presence of cav-1, and the current densities of hHCN4, hKv1.5 and hKir2.1 were also rescued. These results constitute the first evidence of a compensatory role of cav-1 in the heart, justifying the reduced susceptibility of this organ to caveolinopathies.

AIM:Striatin (Strn) is a scaffold protein expressed in cardiomyocytes (CMs) and alteration of its expression are described in various cardiac diseases. However, the alteration underlying its pathogenicity have been poorly investigated. METHODS:We studied the role(s) of cardiac Strn gene (STRN) by comparing the functional properties of CMs, generated from Strn-KO and isogenic WT mouse embryonic stem cell lines. RESULTS:The spontaneous beating rate of Strn-KO CMs was faster than WT cells, and this correlated with a larger fast INa conductance and no changes in If. Paced (2-8 Hz) Strn-KO CMs showed prolonged action potential (AP) duration in comparison with WT CMs and this was not associated with changes in ICaL and IKr. Motion video tracking analysis highlighted an altered contraction in Strn-KO CMs; this was associated with a global increase in intracellular Ca2+, caused by an enhanced late Na+ current density (INaL) and a reduced Na+/Ca2+ exchanger (NCX) activity and expression. Immunofluorescence analysis confirmed the higher Na+ channel expression and a more dynamic microtubule network in Strn-KO CMs than in WT. Indeed, incubation of Strn-KO CMs with the microtubule stabilizer taxol, induced a rescue (downregulation) of INa conductance toward WT levels. CONCLUSION:Loss of STRN alters CMs electrical and contractile profiles and affects cell functionality by a disarrangement of Strn-related multi-protein complexes. This leads to impaired microtubules dynamics and Na+ channels trafficking to the plasma membrane, causing a global Na+ and Ca2+ enhancement.

AIMS:Nfix is a transcription factor belonging to the Nuclear Factor I (NFI) family comprising four members (Nfia, b, c, x). Nfix plays important roles in the development and function of several organs. In muscle development, Nfix controls the switch from embryonic to fetal myogenesis by promoting fast twitching fibres. In the adult muscle, following injury, lack of Nfix impairs regeneration, inducing higher content of slow-twitching fibres. Nfix is expressed also in the heart, but its function has been never investigated before. We studied Nfix role in this organ. METHODS:Using Nfix-null and wild type (WT) mice we analyzed: (1) the expression pattern of Nfix during development by qPCR and (2) the functional alterations caused by its absence, by in vivo telemetry and in vitro patch clamp analysis. RESULTS AND CONCLUSIONS:Nfix expression start in the heart from E12.5. Adult hearts of Nfix-null mice show a hearts morphology and sarcomeric proteins expression similar to WT. However, Nfix-null animals show tachycardia that derives form an intrinsic higher beating rate of the sinus node (SAN). Molecular and functional analysis revealed that sinoatrial cells of Nfix-null mice express a significantly larger L-type calcium current (Cacna1d + Cacna1c). Interestingly, downregulation of Nfix by sh-RNA in primary cultures of neonatal rat ventricular cardiomyocytes induced a similar increase in their spontaneous beating rate and in ICaL current. In conclusion, our data provide the first demonstration of a role of Nfix that, increasing the L-type calcium current, modulates heart rate.

Atrial fibrillation (AF) is the most common cardiac arrhythmia worldwide; however, the underlying causes of AF initiation are still poorly understood, particularly because currently available models do not allow in distinguishing the initial causes from maladaptive remodeling that induces and perpetuates AF. Lately, the genetic background has been proven to be important in the AF onset. iPSC-derived cardiomyocytes, being patient- and mutation-specific, may help solve this diatribe by showing the initial cell-autonomous changes underlying the development of the disease. Transcription factor paired-like homeodomain 2 (PITX2) has been identified as a key regulator of atrial development/differentiation, and the PITX2 genomic locus has the highest association with paroxysmal AF. PITX2 influences mitochondrial activity, and alterations in either its expression or function have been widely associated with AF. In this work, we investigate the activity of mitochondria in iPSC-derived atrial cardiomyocytes (aCMs) obtained from a young patient (24 years old) with paroxysmal AF, carrying a gain-of-function mutation in PITX2 (rs138163892) and from its isogenic control (CTRL) in which the heterozygous point mutation has been reverted to WT. PITX2 aCMs show a higher mitochondrial content, increased mitochondrial activity, and superoxide production under basal conditions when compared to CTRL aCMs. However, increasing mitochondrial workload by FCCP or β-adrenergic stimulation allows us to unmask mitochondrial defects in PITX2 aCMs, which are incapable of responding efficiently to the higher energy demand, determining ATP deficiency.

Abstract Background Anthracyclines effectiveness is burdened by numerous side effects, among which cardiotoxicity (CTX) is the one carrying the highest impact on survival. Prevention of CTX is known to be far more effective than its treatment. Nonetheless, present patients stratification does not predict different post-treatment outcomes. The inter-individual variability in the response to treatment likely resides at the molecular level. In this context, mitochondrial ferritin (FtMt) has been studied in preclinical models of doxorubicin-induced cardiotoxicity. Both in cell lines and animal models, overexpression of this protein has been found to be protective against the cytotoxic oxidative damage deriving from anthracycline use. Aim of the study The primary aim of the study was to evaluate – for the first time in humans and in mouse myocardial cells lines – the expression of FtMt and its relationship with the potential development of cardiotoxicity in patients undergoing chemotherapy with anthracyclines. Methods Twenty-nine patients referred to our Oncology Outpatient Clinic to start treatment with anthracyclines – for either lymphoma or breast cancer – were enrolled before treatment initiation. All patients were above 18 years of age and free from any cardiovascular pathology at baseline. Troponin T (TnT), Brain Natriuretic Peptide (NT-proBNP), FtMt and creatinine were evaluated at baseline, before any chemotherapy cycle, at the end of the protocol, at 6 and 12 months from the first cycle. TnT and NT-proBNP were quantified through ECLIA and the expression of FtMt through qRT-PCR performed on peripheral white blood cells. Left ventricular function was evaluated through standard echocardiography at baseline, at the end of chemotherapy, at 6 and 12 months. Furthermore, variations in FtMt expression in response to doxorubicin treatment were studied on mouse primary cardiomyocytes. Results Direct evaluation of FtMt expression in mouse myocardial cells showed higher levels of FtMt in cardiomyocytes exposed to doxorubicin (p=0.0239). In the clinical model FtMt expression was found to be decreased after treatment initiation, with a trend that in the descriptive analysis appeared to be opposite to the one registered for TnT. For any unit of FtMt at baseline, single TnT values after treatment and at one year were found to be decreased of 1,89 ng/L and of 1,57 ng/L, respectively. TnT was the only cardiac parameter showing significant variation following anthracycline administration, with an average increase of 10 ng/L (p<0,0001) and 15,8 ng/L (p=0,0071) in breast cancer and lymphoma patients respectively and remained elevated at follow-up only in the lymphoma group with a difference of 11,35ng/L at 1 year (p=0,0017). No occurrence of CTX – defined according to international guidelines - was found in the population under study. Conclusions Our results confirm that exposure to anthracyclines influences FtMt expression. The study performed on mouse primary cardiomyocytes demonstrate for the first time the ability of cardiac myocytes to increase FtMt expression in response to doxorubicin. In the clinical model any additional unit of FtMt at baseline was associated with a reduction in TnT values at the end of the treatment and at 1 year, potentially indicating a protective role of this protein in this context. The lack of frank cardiotoxicity in this group impeded the evaluation of the prognostic meaning of this event. In the future drugs able to upregulate the expression of this protein should be investigated as potential strategy to prevent anthracycline-induced cardiotoxicity.

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.

Properties of the funny current ( I f ) have been studied in several animal and cellular models, but so far little is known concerning its properties in human pacemaker cells. This work provides a detailed characterization of I f in human-induced pluripotent stem cell (iPSC)–derived pacemaker cardiomyocytes (pCMs), at different time points. Patch-clamp analysis showed that I f density did not change during differentiation; however, after day 30, it activates at more negative potential and with slower time constants. These changes are accompanied by a slowing in beating rate. I f displayed the voltage-dependent block by caesium and reversed ( E rev ) at − 22 mV, compatibly with the 3:1 K + /Na + permeability ratio. Lowering [Na + ] o (30 mM) shifted the E rev to − 39 mV without affecting conductance. Increasing [K + ] o (30 mM) shifted the E rev to − 15 mV with a fourfold increase in conductance. pCMs express mainly HCN4 and HCN1 together with the accessory subunits CAV3, KCR1, MiRP1, and SAP97 that contribute to the context-dependence of I f . Autonomic agonists modulated the diastolic depolarization, and thus rate, of pCMs. The adrenergic agonist isoproterenol induced rate acceleration and a positive shift of I f voltage-dependence (EC 50 73.4 nM). The muscarinic agonists had opposite effects (Carbachol EC 50 , 11,6 nM). Carbachol effect was however small but it could be increased by pre-stimulation with isoproterenol, indicating low cAMP levels in pCMs. In conclusion, we demonstrated that pCMs display an I f with the physiological properties expected by pacemaker cells and may thus represent a suitable model for studying human I f -related sinus arrhythmias.