Sodium-glucose cotransporter 2 inhibitors (SGLT2i) represent the cornerstone of therapy in patients with type 2 diabetes (T2D), heart failure (HF), or chronic kidney disease (CKD). These patients present a high risk of cardiac arrhythmias, particularly when these comorbidities coexist. In experimental models, SGLT2i exert antiarrhythmic effects and clinical studies and meta-analyses strongly suggest that they reduce new-onset and recurrences of atrial fibrillation in patients with HF or CKD irrespective of the diabetic status. Although some trials and meta-analyses suggest that SGLT2i could decrease the risk of ventricular arrhythmias and sudden cardiac arrest, the evidence is weak, and their potential remains to be confirmed. Thus, clinical evidence so far should be considered as hypothesis-generating. Although the exact mechanism underlying their antiarrhythmic effects remains uncertain and much research is needed, multiple direct cardiac and extracardiac effects may be involved. They improve cardiac electrical (via changes in ion channels and transporters; maintenance of Na+ and Ca2+ homeostasis), structural (reduce hypertrophy, fibrosis, inflammation, and epicardial fat; improve mitochondrial function and energetic metabolism), and autonomic (reduce sympathetic hyperactivity) remodelling. Indirect extracardiac effects related to an improvement in cardiovascular risk factors and haemodynamics, together with their protective renal and vascular effects, may also play a role. This narrative review summarises the experimental and clinical evidence of their antiarrhythmic effects, potential underlying mechanisms, limitations of present evidence, and gaps of knowledge that should be filled before SGLT2i can be recommended for the prevention and treatment of arrhythmias in patients for whom these drugs are indicated.

Propionic acidemia (PA) is a metabolic disorder caused by a deficiency of the mitochondrial enzyme propionyl-CoA carboxylase (PCC) due to mutations in the PCCA or PCCB genes, which encode the two PCC subunits. PA may lead to several types of cardiomyopathy and has been linked to cardiac electrical abnormalities such as QT interval prolongation, life-threatening arrhythmias, and sudden cardiac death. To gain insights into the mechanisms underlying PA-induced proarrhythmia, we recorded action potentials (APs) and ion currents using whole-cell patch-clamp in ventricular-like induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) from a PA patient carrying two pathogenic mutations in the PCCA gene (p.Cys616_Val633del and p.Gly477Glufs*9) (PCCA cells) and from a healthy subject (healthy cells). In cells driven at 1 Hz, PCC deficiency increased the latency and prolonged the AP duration (APD) measured at 20% of repolarization, without modifying resting membrane potential or AP amplitude. Moreover, delayed afterdepolarizations appeared at the end of the repolarization phase in unstimulated and paced PCCA cells. PCC deficiency significantly reduced peak sodium current (INa) but increased the late INa (INaL) component. In addition, L-type Ca2+ current (ICaL) density was reduced, while the inward and outward density of the Na+/Ca2+ exchanger current (INCX) was increased in PCCA cells compared to healthy ones. In conclusion, our results demonstrate that at the cellular level, PCC deficiency can modify the ion currents controlling cardiac excitability, APD, and intracellular Ca2+ handling, increasing the risk of arrhythmias independently of the progressive late-onset cardiomyopathy induced by PA disease.

Mutations in MINDBOMB 1 ( MIB1 ), encoding an E3 ubiquitin ligase of the NOTCH signaling pathway, cause left ventricular noncompaction cardiomyopathy (LVNC) in mice and humans, increasing the risk of arrhythmia and left ventricular dysfunction. This study aimed to investigate the effect of MIB1 mutations on cardiac electrical activity. We examined male Mib1 flox ;Tnnt2 Cre mice, a disease model of LVNC, and wildtype littermates on the C57BL/6J genetic background. Our results demonstrate that the gap-junction protein connexin43 was delocalized from the intercalated disks to the lateral long axis of Mib1 flox ;Tnnt2 Cre cardiomyocytes. Cardiomyocyte electrophysiology revealed an increase in the Na (I Na ) peak density at potentials between -50 and -30 mV in Mib1 flox ;Tnnt2 Cre mice, with no changes in I Na activation or inactivation kinetics. Mib1 flox ;Tnnt2 Cre cardiomyocytes also showed decreases in outward K + peak currents and currents at the end of depolarizing pulses at potentials ≥−10 mV and ≥−20 mV, respectively, and this was accompanied by a lower charge density at ≥−20 mV. Action potential duration was increased in Mib1 flox ;Tnnt2 Cre cardiomyocytes. The cardiac stress, induced by swimming endurance training or β-adrenergic stimulation with isoproterenol, increases QTc duration in Mib1 flox ;Tnnt2 Cre mice, accompanied by a decrease in T-wave amplitude and area. Swimming endurance training decreased heart rate in wildtype and Mib1 flox ;Tnnt2 Cre mice but was unaffected by long-term isoproterenol treatment. These mouse findings are in agreement with an increased QTc duration found in LVNC patients carrying MIB1 mutations. These results provide insight into the outcomes of LVNC and relate its pathogenicity to impaired ventricular repolarization.

Dapagliflozin and empagliflozin exert many cardiovascular protective actions in heart failure (HF) patients. HF-induced electrical remodelling decreases the expression of Nav1.5 channels (encoded by SCN5A) that generate the cardiac Na+ current (INa) impairing excitability and promoting arrhythmias. We aimed to mechanistically decipher the peak INa increase produced by dapagliflozin and empagliflozin in healthy and HF cardiomyocytes. We recorded macroscopic and single-channel currents and action potentials (AP) using the patch-clamp technique and generated a mouse model of HF with reduced ejection fraction by transverse aortic constriction (TAC). Single-channel recordings showed that dapagliflozin and empagliflozin (1 μM) increased the open probability (Po) of Nav1.5 channels by augmenting channel re-openings and the number of traces with openings and by doubling the open time constant, respectively. Both drugs increased SCN5A mRNA levels and the membrane expression of Nav1.5 channels. Empagliflozin also enhanced the cytoplasmic mobility of Nav1.5 channels. Molecular modelling and site-directed mutagenesis analysis demonstrated that both drugs bind to a previously unknown site at the Nav1.5 DIII-DIV fenestration. Dapagliflozin and empagliflozin hyperpolarized the resting membrane potential and increased the action potential amplitude in human cardiomyocytes derived from induced pluripotent stem cells. Importantly, in TAC cardiomyocytes dapagliflozin and empagliflozin restored the HF-reduced peak INa to control levels. Dapagliflozin and empagliflozin bind to a novel site within cardiac Nav1.5 increasing INa by augmenting the Po and the membrane expression of the channels. We hypothesized that this unique effects could be of interest for the treatment of arrhythmias associated with decreased Nav1.5 channel expression.

Atrial fibrillation (AF) is the most common cardiac arrhythmia and is associated with an increased morbidity and mortality. There is clinical evidence that an increasing number of cardiovascular and non-cardiovascular drugs, mainly anticancer drugs, can induce AF either in patients with or without pre-existing cardiac disorders, but drug-induced AF (DIAF) has not received the attention that it might deserve. In many cases DIAF is asymptomatic and paroxysmal and patients recover sinus rhythm spontaneously, but sometimes, DIAF persists, and it is necessary to perform a cardioversion. Furthermore, DIAF is not mentioned in clinical guidelines on the treatment of AF. The risk of DIAF increases in elderly and in patients treated with polypharmacy and with risk factors and comorbidities that commonly coexist with AF. This is the case of cancer patients. Under these circumstances ascribing causality of DIAF to a given drug often represents a clinical challenge. We review the incidence, the pathophysiological mechanisms, risk factors, clinical relevance, and treatment of DIAF. Because of the limited information presently available, further research is needed to obtain a deeper insight into DIAF. Meanwhile, it is important that clinicians are aware of the problem that DIAF represents, recognize which drugs may cause DIAF, and consider the possibility that a drug may be responsible for a new-onset AF episode.

BACKGROUND: Adipocyte FABP4 (fatty acid-binding protein 4) is augmented in the epicardial stroma of patients with long-standing persistent atrial fibrillation. Because this molecule is released mainly by adipocytes, our objective was to study its role in atrial cardiomyopathy, focusing our attention on fibrosis, metabolism, and electrophysiological changes. These results might clarify the role of adiposity as a mediator of atrial cardiomyopathy. METHODS: We used several preclinical cellular models, epicardial and subcutaneous stroma primary cell cultures from patients undergoing open heart surgery, human atrial fibroblasts, atrial cardiomyocytes derived from human induced pluripotent stem cells and isolated from adult mice, and Nav1.5 transfected Chinese hamster ovary cells. Fibrosis, glucose, mitochondrial and adipogenesis activity, gene expression, and proteomics were determined by wound healing, enzymatic, colorimetric, fluorescence assays, real-time quantitative polymerase chain reaction, and TripleTOF proteomics. Molecular changes were analyzed by Raman confocal microspectroscopy, calcium dynamics by confocal microscopy, and ion currents by patch clamp. Epicardial, subcutaneous, and atrial fibroblasts and cardiomyocytes were incubated with FABP4 at 100 ng/mL. RESULTS: Our results showed that FABP4 induced fibrosis, glucose metabolism, and lipid accumulation on epicardial and subcutaneous stroma cells and atrial fibroblasts. Besides, it modified lipid content and calcium dynamics in atrial cardiomyocytes without effects on I Na . CONCLUSIONS: FABP4 exerts fibrotic and metabolic changes on epicardial stroma and modifies lipid content and calcium dynamic on atrial cardiomyocytes. These results suggest its possible role as an atrial cardiomyopathy mediator.

A novel rare mutation in the pore region of Nav1.5 channels (p.L889V) has been found in three unrelated Spanish families that produces quite diverse phenotypic manifestations (Brugada syndrome, conduction disease, dilated cardiomyopathy, sinus node dysfunction, etc.) with variable penetrance among families. We clinically characterized the carriers and recorded the Na+ current (INa) generated by p.L889V and native (WT) Nav1.5 channels, alone or in combination, to obtain further insight into the genotypic–phenotypic relationships in patients carrying SCN5A mutations and in the molecular determinants of the Nav1.5 channel function. The variant produced a strong dominant negative effect (DNE) since the peak INa generated by p.L889V channels expressed in Chinese hamster ovary cells, either alone (−69.4 ± 9.0 pA/pF) or in combination with WT (−62.2 ± 14.6 pA/pF), was significantly (n ≥ 17, p < 0.05) reduced compared to that generated by WT channels alone (−199.1 ± 44.1 pA/pF). The mutation shifted the voltage dependence of channel activation and inactivation to depolarized potentials, did not modify the density of the late component of INa, slightly decreased the peak window current, accelerated the recovery from fast and slow inactivation, and slowed the induction kinetics of slow inactivation, decreasing the fraction of channels entering this inactivated state. The membrane expression of p.L889V channels was low, and in silico molecular experiments demonstrated profound alterations in the disposition of the pore region of the mutated channels. Despite the mutation producing a marked DNE and reduction in the INa and being located in a critical domain of the channel, its penetrance and expressivity are quite variable among the carriers. Our results reinforce the argument that the incomplete penetrance and phenotypic variability of SCN5A loss-of-function mutations are the result of a combination of multiple factors, making it difficult to predict their expressivity in the carriers despite the combination of clinical, genetic, and functional studies.

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BackgroundFamilial association of atrial fibrillation (AF) can involve single gene variants related to known arrhythmogenic mechanisms; however, genome-wide association studies often disclose complex genetic variants in familial and non-familial AF, making it difficult to relate to known pathogenetic mechanisms.MethodsThe finding of 4 siblings with AF led to studying 47 members of a family. Long-term Holter monitoring (298 hours average) ruled out silent AFWhole-exome sequencing was performed and variants shared by the index cases were filtered and prioritized according to current recommendations. HCN4 currents (IHCN4) were recorded in Chinese hamster ovary cells expressing human p.P1163H and/or native Hcn4 channels using the patch-clamp technique and topologically associated domain analysis of GATA5 variant carriers were performed.ResultsThe clinical study diagnosed 2 more AF cases. Five family members carried the heterozygous p.P1163H, HCN4 variant, 14 the intronic 20,61040536,G,A GATA5 rare variant, and 9 carried both variants (HCN4+GATA5). Five of the 6 AF cases (onset age ranging 33-70 years) carried both variants and one the GATA5 variant alone. Multivariate analysis showed that the presence of HCN4+GATA5 variants significantly and independently increased AF risk [OR=32.740 (1.812-591.408)] and not age, hypertension or overweight. Functional testing showed that IHcn4 generated by heterozygous p.P1163H were normal. Topologically associating domain analysis suggested that GATA5 could affect the expression of many genes, including those encoding microRNA-1.ConclusionThe coincidence of two rare gene variants was independently associated with AF, but functional studies do not allow the postulation of the arrhythmogenic mechanism(s) involved.

ABSTRACTBackgroundIn a family with inappropriate sinus tachycardia (IST) we identified a novel mutation (p.V240M) of the hyperpolarization-activated cyclic nucleotide-gated type 4 (HCN4) channel, which contributes to the pacemaker current (If) in human sinoatrial node cells. Here we clinically study the family and functionally analyze the p.V240M variant.MethodsMacroscopic (IHCN4) and single-channel currents were recorded using patch-clamp in cells expressing human native (WT) and/or p.V240M HCN4 channels.ResultsAll p.V240M mutation carriers exhibited IST (mean heart rate 113[7] bpm, n=9), that in adults, was accompanied by cardiomyopathy. IHCN4generated by p.V240M channels either alone or in combination with WT was significantly greater than that generated by WT channels. The variant, which lies in the N-terminal HCN domain, increased single-channel conductance and opening frequency and probability of HCN4 channels. Conversely, it did not modify channel sensitivity for cAMP and ivabradine or the level of expression at the membrane. Treatment with ivabradine based on functional data reversed the IST and the cardiomyopathy of the carriers.ConclusionsThe p.V240M gain-of-function variant increases Ifduring diastole, which explains the IST of the carriers. The results demonstrate the importance of the unique HCN domain in HCN4 which stabilizes the channels in the closed state.FundingMinisterio de Ciencia e Innovación (PID2020-118694RB-I00); Comunidad Autónoma de Madrid (P2022/BMD-7229), European Structural and Investment Funds); and Instituto de Salud Carlos III (CIBERCV; CB16/11/00303).

INTRODUCTION:Arterial hypertension represents the leading modifiable risk factor for all-cause death and early development of cardiovascular disease in women. Current clinical guidelines for the treatment of hypertension noted that women respond to antihypertensive drugs similarly to men and, therefore, treatment recommendations remain the same for both sexes. However, clinical evidence suggests the existence of sex- and gender-related differences (SGRD) in the prevalence, pathophysiology, pharmacodynamics (efficacy and safety) and pharmacokinetics of antihypertensive drugs.AREAS COVERED:This review summarizes SGRD in the prevalence of hypertension, hypertension-mediated organ damage and blood pressure control, prescription patterns, and pharmacokinetics/ pharmacodynamics and doses of antihypertensive drugs.EXPERT OPINION:There is limited information on SGRD in antihypertensive drug efficacy because of the underrepresentation of women in randomized clinical trials and, more important, because few trials reported results stratified by sex or performed sex-specific analyses. However, there are SGRD in hypertension-mediated organ damage, drug pharmacokinetics and, particularly, in drug safety. Prospective trials specifically designed to better understand the basis for SGRD in the pathophysiology of hypertension and in the efficacy and safety of antihypertensive drugs are needed to achieve a more personalized treatment of hypertension and hypertension-mediated organ damage in women.

In a family with inappropriate sinus tachycardia (IST), we identified a mutation (p.V240M) of the hyperpolarization-activated cyclic nucleotide-gated type 4 (HCN4) channel, which contributes to the pacemaker current (If) in human sinoatrial node cells. Here, we clinically study fifteen family members and functionally analyze the p.V240M variant. Macroscopic (IHCN4) and single-channel currents were recorded using patch-clamp in cells expressing human native (WT) and/or p.V240M HCN4 channels. All p.V240M mutation carriers exhibited IST that was accompanied by cardiomyopathy in adults. IHCN4 generated by p.V240M channels either alone or in combination with WT was significantly greater than that generated by WT channels alone. The variant, which lies in the N-terminal HCN domain, increased the single-channel conductance and opening frequency and probability of HCN4 channels. Conversely, it did not modify the channel sensitivity for cAMP and ivabradine or the level of expression at the membrane. Treatment with ivabradine based on functional data reversed the IST and the cardiomyopathy of the carriers. In computer simulations, the p.V240M gain-of-function variant increases If and beating rate and thus explains the IST of the carriers. The results demonstrate the importance of the unique HCN domain in HCN4, which stabilizes the channels in the closed state.

Cardiac electrical activity is governed by different ion channels that generate action potentials. Acquired or inherited abnormalities in the expression and/or function of ion channels usually result in electrophysiological changes that can cause cardiac arrhythmias. Transcription factors (TFs) control gene transcription by binding to specific DNA sequences adjacent to target genes. Linkage analysis, candidate-gene screening within families, and genome-wide association studies have linked rare and common genetic variants in the genes encoding TFs with genetically-determined cardiac arrhythmias. Besides its critical role in cardiac development, recent data demonstrated that they control cardiac electrical activity through the direct regulation of the expression and function of cardiac ion channels in adult hearts. This narrative review summarizes some studies showing functional data on regulation of the main human atrial and ventricular Na+, Ca2+, and K+ channels by cardiac TFs such as Pitx2c, Tbx20, Tbx5, Zfhx3, among others. The results have improved our understanding of the mechanisms regulating cardiac electrical activity and may open new avenues for therapeutic interventions in cardiac acquired or inherited arrhythmias through the identification of TFs as potential drug targets. Even though TFs have for a long time been considered as 'undruggable' targets, advances in structural biology have led to the identification of unique pockets in TFs amenable to be targeted with small-molecule drugs or peptides that are emerging as novel therapeutic drugs.

Abstract Background Empagliflozin (EMPA) and dapagliflozin (DAPA) are sodium-glucose cotransporter 2 inhibitors (SGLT2i) used for the treatment of type 2 Diabetes Mellitus (T2DM). Both drugs reduce morbidity and mortality in heart failure (HF) patients with reduced or preserved ejection fraction, even in the absence of T2DM. Moreover, these drugs decrease ventricular arrhythmias and sudden cardiac death in HF patients. The sodium current (INa), carried by Nav1.5 channels, is responsible for cardiac action potential (AP) depolarization and determines excitability and conduction velocity. In HF patients, the expression of Nav1.5 channels is reduced, leading to a decrease of ventricular excitability that enhances the arrhythmic risk. Purpose We aimed to determine the effects of EMPA and DAPA on human cardiac INa and AP characteristics. Methods Peak INa and ventricular-like APs were recorded in cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) using patch-clamp techniques. INa was also recorded in CHO cells transiently transfected with human Nav1.5+Navβ1 channels. In all cases, EMPA or DAPA (1 μM) were added to culture media and incubated for 24-h. Results APs recorded in hiPSC-CMs exhibited automatic activity and incubation with EMPA or DAPA did not modify spontaneous beating frequency (0.39±0.04 Hz; P>0.05, n≥16). In cells driven at 1 Hz, none of the drugs modified resting membrane potential (−76.7±1.4 mV; P>0.05, n≥11), but significantly increased AP amplitude from 98.6±3.6 to 105±2.2 (DAPA) and 107±2.3 mV (EMPA) (P<0.05). Interestingly, only EMPA lengthened AP duration measured at 20%, 50%, and 90% (from 605.6±31.3 to 760.5±59.0 ms, P<0.05) of repolarization. In hiPSC-CMs EMPA increased maximum INa density by 64% (from −156.0±28.0 to −256.4±28.1 pA/pF, P<0.05, n≥7) and shifted the midpoint (Vh) of the inactivation curve to more hyperpolarized potentials (from −97.3±4.5 to −108.6±4.4 mV, P<0.05, n≥7). In turn, DAPA increased maximum INa density by 24% (to −193.8±26.6 pA/pF) and shifted the Vh of the activation curve to more negative potentials (from −47.2±1.6 mV to −55.5±2.8 mV, P<0.05), an effect that would increase the INa at negative potentials coinciding with channel opening. None of the drugs modified the time course of current activation or inactivation. In CHO cells, EMPA and DAPA effects on INa were identical to those observed on hiPSC-CM. These results suggest that both SGLT2i increase INa by enhancing Nav1.5 expression into the cell membrane, by a direct gating effect on the channel, or by a combination of both. Conclusions In human cardiomyocytes, EMPA and DAPA increase INa and the AP amplitude. Moreover, EMPA, but not DAPA, prolonged AP duration. We propose that EMPA and DAPA exhibit a unique mechanism that increases cardiac excitability and conduction velocity and could contribute to the prevention of arrhythmic events in HF patients. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministerio de Ciencia e innovaciόnInstituto de de Salud Carlos III