Coupling and heterogeneity modulate pacemaking capability in healthy and diseased two-dimensional sinoatrial node tissue models.

Both experimental and modeling studies have attempted to determine mechanisms by which a small anatomical region, such as the sinoatrial node (SAN), can robustly drive electrical activity in the human heart. However, despite many advances from prior research, important questions remain unanswered. This study aimed to investigate, through mathematical modeling, the roles of intercellular coupling and cellular heterogeneity in synchronization and pacemaking within the healthy and diseased SAN. In a multicellular computational model of a monolayer of either human or rabbit SAN cells, simulations revealed that heterogenous cells synchronize their discharge frequency into a unique beating rhythm across a wide range of heterogeneity and intercellular coupling values. However, an unanticipated behavior appeared under pathological conditions where perturbation of ionic currents led to reduced excitability. Under these conditions, an intermediate range of intercellular coupling (900-4000 MΩ) was beneficial to SAN automaticity, enabling a very small portion of tissue (3.4%) to drive propagation, with propagation failure occurring at both lower and higher resistances. This protective effect of intercellular coupling and heterogeneity, seen in both human and rabbit tissues, highlights the remarkable resilience of the SAN. Overall, the model presented in this work allowed insight into how spontaneous beating of the SAN tissue may be preserved in the face of perturbations that can cause individual cells to lose automaticity. The simulations suggest that certain degrees of gap junctional coupling protect the SAN from ionic perturbations that can be caused by drugs or mutations.

The SPTLC1 p.S331 mutation bridges sensory neuropathy and motor neuron disease and has implications for treatment.

AIMS: SPTLC1-related disorder is a late onset sensory-autonomic neuropathy associated with perturbed sphingolipid homeostasis which can be improved by supplementation with the serine palmitoyl-CoA transferase (SPT) substrate, l-serine. Recently, a juvenile form of motor neuron disease has been linked to SPTLC1 variants. Variants affecting the p.S331 residue of SPTLC1 cause a distinct phenotype, whose pathogenic basis has not been established. This study aims to define the neuropathological and biochemical consequences of the SPTLC1 p.S331 variant, and test response to l-serine in this specific genotype. METHODS: We report clinical and neurophysiological characterisation of two unrelated children carrying distinct p.S331 SPTLC1 variants. The neuropathology was investigated by analysis of sural nerve and skin innervation. To clarify the biochemical consequences of the p.S331 variant, we performed sphingolipidomic profiling of serum and skin fibroblasts. We also tested the effect of l-serine supplementation in skin fibroblasts of patients with p.S331 mutations. RESULTS: In both patients, we recognised an early onset phenotype with prevalent progressive motor neuron disease. Neuropathology showed severe damage to the sensory and autonomic systems. Sphingolipidomic analysis showed the coexistence of neurotoxic deoxy-sphingolipids with an excess of canonical products of the SPT enzyme. l-serine supplementation in patient fibroblasts reduced production of toxic 1-deoxysphingolipids but further increased the overproduction of sphingolipids. CONCLUSIONS: Our findings suggest that p.S331 SPTLC1 variants lead to an overlap phenotype combining features of sensory and motor neuropathies, thus proposing a continuum in the spectrum of SPTLC1-related disorders. l-serine supplementation in these patients may be detrimental.

Inflammation as a Risk Factor in Cardiotoxicity: An Important Consideration for Screening During Drug Development.

Numerous commonly prescribed drugs, including antiarrhythmics, antihistamines, and antibiotics, carry a proarrhythmic risk and may induce dangerous arrhythmias, including the potentially fatal Torsades de Pointes. For this reason, cardiotoxicity testing has become essential in drug development and a required step in the approval of any medication for use in humans. Blockade of the hERG K+ channel and the consequent prolongation of the QT interval on the ECG have been considered the gold standard to predict the arrhythmogenic risk of drugs. In recent years, however, preclinical safety pharmacology has begun to adopt a more integrative approach that incorporates mathematical modeling and considers the effects of drugs on multiple ion channels. Despite these advances, early stage drug screening research only evaluates QT prolongation in experimental and computational models that represent healthy individuals. We suggest here that integrating disease modeling with cardiotoxicity testing can improve drug risk stratification by predicting how disease processes and additional comorbidities may influence the risks posed by specific drugs. In particular, chronic systemic inflammation, a condition associated with many diseases, affects heart function and can exacerbate medications' cardiotoxic effects. We discuss emerging research implicating the role of inflammation in cardiac electrophysiology, and we offer a perspective on how in silico modeling of inflammation may lead to improved evaluation of the proarrhythmic risk of drugs at their early stage of development.

In vitro and In silico Models to Study SARS-CoV-2 Infection: Integrating Experimental and Computational Tools to Mimic "COVID-19 Cardiomyocyte".

The rapid dissemination of SARS-CoV-2 has made COVID-19 a tremendous social, economic, and health burden. Despite the efforts to understand the virus and treat the disease, many questions remain unanswered about COVID-19 mechanisms of infection and progression. Severe Acute Respiratory Syndrome (SARS) infection can affect several organs in the body including the heart, which can result in thromboembolism, myocardial injury, acute coronary syndromes, and arrhythmias. Numerous cardiac adverse events, from cardiomyocyte death to secondary effects caused by exaggerated immunological response against the virus, have been clinically reported. In addition to the disease itself, repurposing of treatments by using "off label" drugs can also contribute to cardiotoxicity. Over the past several decades, animal models and more recently, stem cell-derived cardiomyocytes have been proposed for studying diseases and testing treatments in vitro. In addition, mechanistic in silico models have been widely used for disease and drug studies. In these models, several characteristics such as gender, electrolyte imbalance, and comorbidities can be implemented to study pathophysiology of cardiac diseases and to predict cardiotoxicity of drug treatments. In this Mini Review, we (1) present the state of the art of in vitro and in silico cardiomyocyte modeling currently in use to study COVID-19, (2) review in vitro and in silico models that can be adopted to mimic the effects of SARS-CoV-2 infection on cardiac function, and (3) provide a perspective on how to combine some of these models to mimic "COVID-19 cardiomyocytes environment.".

Canakinumab as treatment for COVID-19-related pneumonia: A prospective case-control study.

OBJECTIVES: Canakinumab is an IL-1ß antibody that neutralises the activity of IL-1ß. This study examined the efficacy and safety of canakinumab in patients with moderate COVID-19-related pneumonia. DESIGN: This study aimed to evaluate the reduction in duration of hospitalisation with adequate oxygen status. Forty-eight patients with moderate COVID-19-related pneumonia were asked to participate in the prospective case-control study: 33 patients (cases) signed informed consent and received canakinumab (Cohort 1) and 15 patients (Controls) refused to receive the experimental drug and received institutional standard of care (Cohort 2). RESULTS: Hospital discharge within 21 days was seen in 63% of patients in Cohort 1 vs. 0% in Cohort 2 (median 14 vs. 26 days, respectively; p < 0.001). There was significant clinical improvement in ventilation regimes following administration of canakinumab compared with Cohort 2 (Stuart-Maxwell test for paired data, p < 0.001). Patients treated with canakinumab experienced a significant increase in PaO2:FiO2 (p < 0.001) and reduction in lung damage by CT (p = 0.01), along with significant decreases in immune/inflammation markers that were not observed in Cohort 2. Only mild side-effects were seen in patients treated with canakinumab; survival at 60 days was 90.0% (95% CI 71.9-96.7) in patients treated with canakinumab and 73.3% (95% CI 43.6-89.1) for Cohort 2. CONCLUSIONS: Treatment with canakinumab in patients with COVID-19-related pneumonia rapidly restored normal oxygen status, decreased the need for invasive mechanical ventilation, and was associated with earlier hospital discharge and favourable prognosis versus standard of care.

Investigational Treatments for COVID-19 may Increase Ventricular Arrhythmia Risk Through Drug Interactions.

Many drugs that have been proposed for treatment of coronavirus disease 2019 (COVID-19) are reported to cause cardiac adverse events, including ventricular arrhythmias. In order to properly weigh risks against potential benefits, particularly when decisions must be made quickly, mathematical modeling of both drug disposition and drug action can be useful for predicting patient response and making informed decisions. Here, we explored the potential effects on cardiac electrophysiology of four drugs proposed to treat COVID-19: lopinavir, ritonavir, chloroquine, and azithromycin, as well as combination therapy involving these drugs. Our study combined simulations of pharmacokinetics (PKs) with quantitative systems pharmacology (QSP) modeling of ventricular myocytes to predict potential cardiac adverse events caused by these treatments. Simulation results predicted that drug combinations can lead to greater cellular action potential prolongation, analogous to QT prolongation, compared with drugs given in isolation. The combination effect can result from both PK and pharmacodynamic drug interactions. Importantly, simulations of different patient groups predicted that women with pre-existing heart disease are especially susceptible to drug-induced arrhythmias, compared with diseased men or healthy individuals of either sex. Statistical analysis of population simulations revealed the molecular factors that make certain women with heart failure especially susceptible to arrhythmias. Overall, the results illustrate how PK and QSP modeling may be combined to more precisely predict cardiac risks of COVID-19 therapies.

Investigational treatments for COVID-19 may increase ventricular arrhythmia risk through drug interactions.

Many drugs that have been proposed for treatment of COVID-19 are reported to cause cardiac adverse events, including ventricular arrhythmias. In order to properly weigh risks against potential benefits, particularly when decisions must be made quickly, mathematical modeling of both drug disposition and drug action can be useful for predicting patient response and making informed decisions. Here we explored the potential effects on cardiac electrophysiology of 4 drugs proposed to treat COVID-19: lopinavir, ritonavir, chloroquine, and azithromycin, as well as combination therapy involving these drugs. Our study combined simulations of pharmacokinetics (PK) with quantitative systems pharmacology (QSP) modeling of ventricular myocytes to predict potential cardiac adverse events caused by these treatments. Simulation results predicted that drug combinations can lead to greater cellular action potential prolongation, analogous to QT prolongation, compared with drugs given in isolation. The combination effect can result from both pharmacokinetic and pharmacodynamic drug interactions. Importantly, simulations of different patient groups predicted that females with pre-existing heart disease are especially susceptible to drug-induced arrhythmias, compared males with disease or healthy individuals of either sex. Overall, the results illustrate how PK and QSP modeling may be combined to more precisely predict cardiac risks of COVID-19 therapies.

A distinct molecular mechanism by which phenytoin rescues a novel long QT 3 variant.

BACKGROUND: Genetic variants in SCN5A can result in channelopathies such as the long QT syndrome type 3 (LQT3), but the therapeutic response to Na+ channel blockers can vary. We previously reported a case of an infant with malignant LQT3 and a missense Q1475P SCN5A variant, who was effectively treated with phenytoin, but only partially with mexiletine. Here, we functionally characterized this variant and investigated possible mechanisms for the differential drug actions. METHODS: Wild-type or mutant Nav1.5 cDNAs were examined in transfected HEK293 cells with patch clamping and biochemical assays. We used computational modeling to provide insights into altered channel kinetics and to predict effects on the action potential. RESULTS: The Q1475P variant in Nav1.5 reduced the current density and channel surface expression, characteristic of a trafficking defect. The variant also led to positive shifts in the voltage dependence of steady-state activation and inactivation, faster inactivation and recovery from inactivation, and increased the "late" Na+ current. Simulations of Nav1.5 gating with a 9-state Markov model suggested that transitions from inactivated to closed states were accelerated in Q1475P channels, leading to accumulation of channels in non-inactivated closed states. Simulations with a human ventricular myocyte model predicted action potential prolongation with Q1475P, compared with wild type, channels. Patch clamp data showed that mexiletine and phenytoin similarly rescued some of the gating defects. Chronic incubation with mexiletine, but not phenytoin, rescued the Nav1.5-Q1475P trafficking defect, thus increasing mutant channel expression. CONCLUSIONS: The gain-of-function effects of Nav1.5-Q1475P predominate to cause a malignant long QT phenotype. Phenytoin partially corrects the gating defect without restoring surface expression of the mutant channel, whereas mexiletine restores surface expression of the mutant channel, which may explain the lack of efficacy of mexiletine when compared to phenytoin. Our data makes a case for experimental studies before embarking on a one-for-all therapy of arrhythmias.

Multiscale Functional Clustering Reveals Frequency Dependent Brain Organization in Type II Focal Cortical Dysplasia With Sleep Hypermotor Epilepsy.

OBJECTIVE: A multiscale functional clustering approach is proposed to investigate the organization of the epileptic networks during different sleep stages and in relation with the occurrence of seizures. METHOD: Stereo-electroencephalographic signals from seven pharmaco-resistant epileptic patients (focal cortical dysplasia type II) were analyzed. The discrete wavelet transform provided a multiscale framework on which a data-driven functional clustering procedure was applied, based on multivariate measures of integration and mutual information. The most interacting functional clusters (FCs) within the sampled brain areas were extracted. RESULTS: FCs characterized by strongly integrated activity were observed mostly in the beta and alpha frequency bands, immediately before seizure onset and in deep sleep stages. These FCs generally included the electrodes from the epileptogenic zone. Furthermore, repeatable patterns were found across ictal events in the same patient. CONCLUSION: In line with previous studies, our findings provide evidence of the important role of beta and alpha activity in seizures generation and support the relation between epileptic activity and sleep stages. SIGNIFICANCE: Despite the small number of subjects included in the study, the present results suggest that the proposed multiscale functional clustering approach is a useful tool for the identification of the frequency-dependent mechanisms underlying seizure generation.

Optical Action Potential Mapping in Acute Models of Ischemia-Reperfusion Injury: Probing the Arrhythmogenic Role of the Mitochondrial Translocator Protein.

Ischemia-reperfusion (I/R) injury causes dynamic changes in electrophysiological properties that promote the incidence of post-ischemic arrhythmias. High-resolution optical action potential mapping allows for a quantitative assessment of the electrophysiological substrate at a cellular resolution within the intact heart, which is critical for elucidation of arrhythmia mechanisms. We and others have found that pharmacological inhibition of the translocator protein (TSPO) is highly effective against postischemic arrhythmias. A major hurdle that has limited the translation of this approach to patients is the fact that available TSPO ligands have several confounding effects, including a potent negative ionotropic property. To circumvent such limitations we developed an in vivo cardiac specific TSPO gene silencing approach as an alternative. Here, we provide the methodological details of our optical action potential mapping studies that were designed to probe the effects of TSPO silencing in hearts from spontaneously hypertensive rats (SHR) that are prone to I/R injury.

Calcium metabolism serum markers in adult patients with epilepsy and the effect of vitamin D supplementation on seizure control.

PURPOSE: To evaluate serum markers of calcium metabolism in adult patients with epilepsy (PWE) treated with antiepileptic drugs (AEDs) and the effect of vitamin D supplementation on seizure frequency. METHODS: Serum levels of calcium, phosphate, intact parathyroid hormone (iPTH) and 25-hydroxyvitamin D (25[OH]D) were compared in 160 PWE on chronic therapy with AEDs and 42 matched controls. Blood concentrations were analyzed taking into account the different features of epilepsy and treatment. Finally, the effect of vitamin D supplementation on seizure control was assessed in a subgroup of 48 drug resistant epileptic patients. RESULTS: PWE showed lower serum levels of 25[OH]D compared to control subjects (p < .001). Only 25% PWE showed normal 25[OH]D levels, whereas 41,9% had a vitamin D failure and 33,1% a vitamin D deficiency (p < .001). 25[OH]D serum levels depended on treatment duration, number of medications and enzyme-inducing AEDs (p < .001, p < .001, p = .013, respectively). Polytherapy and enzyme-inducing AEDs showed more detrimental effects on the 25[OH]D and calcium serum levels. The administration of vitamin D failed to significantly improve seizure control. CONCLUSIONS: PWE show deficiency of vitamin D. The serum levels of 25[OH]D depend on the features and duration of AEDs treatment. Vitamin D administration in drug resistant epilepsy patients does not result in a reduction of seizure frequency.

Continuous positive airway pressure treatment with nasal pillows in obstructive sleep apnea: long-term effectiveness and adherence.

BACKGROUNDS: Mask-related side effects can negatively influence adherence to continuous positive airway pressure (CPAP). Nasal pillows (P) can be an alternative to the standard nasal masks (N), although there are no data about their long-term efficacy. This study aimed to assess long-term effectiveness and adherence to CPAP therapy delivered with nasal pillows in obstructive sleep apnea syndrome (OSAS) patients. METHODS: A retrospective observational design involving a series of consecutive CPAP-naïve patients affected by OSAS. After an initial mask fitting session all patients were allowed to choose the type of nasal interface (N or P) they preferred. Outcomes were assessed 5 days after CPAP titration, and after 2 and 12 months. Patients were offered the option of switching to an alternative mask if needed. RESULTS: Data from 144 patients were analyzed. Subjects were predominantly male (76%), middle aged (58.14 ± 12.86), moderately obese (body mass index: 33.89 ± 7.56), and affected by severe OSAS (apnea-hypopnea index: 47.60 ± 21.31). A total of 102 patients (70.8%) chose P, and 42 (29.2%) chose N. Clinical and polygraphic features, and CPAP pressure levels were similar in P and N groups, both at baseline and at 12 months. A good adherence to treatment was observed in both groups (P, 5.5 ± 1.8 h; N, 5.3 ± 1.5 h). Seventy-six patients (53%) reported at least one side effect during the whole study period, without statistically significant between-group differences. Nostril pain was the most frequent side effect in P. CONCLUSIONS: Nasal pillows showed equal long-term effectiveness and objective adherence as standard nasal masks.

Inverse remodelling of K2P3.1 K+ channel expression and action potential duration in left ventricular dysfunction and atrial fibrillation: implications for patient-specific antiarrhythmic drug therapy.

AIMS: Atrial fibrillation (AF) prevalence increases with advanced stages of left ventricular (LV) dysfunction. Remote proarrhythmic effects of ventricular dysfunction on atrial electrophysiology remain incompletely understood. We hypothesized that repolarizing K2P3.1 K+ channels, previously implicated in AF pathophysiology, may contribute to shaping the atrial action potential (AP), forming a specific electrical substrate with LV dysfunction that might represent a target for personalized antiarrhythmic therapy. METHODS AND RESULTS: A total of 175 patients exhibiting different stages of LV dysfunction were included. Ion channel expression was quantified by real-time polymerase chain reaction and Western blot. Membrane currents and APs were recorded from atrial cardiomyocytes using the patch-clamp technique. Severely reduced LV function was associated with decreased atrial K2P3.1 expression in sinus rhythm patients. In contrast, chronic (c)AF resulted in increased K2P3.1 levels, but paroxysmal (p)AF was not linked to significant K2P3.1 remodelling. LV dysfunction-related suppression of K2P3.1 currents prolonged atrial AP duration (APD) compared with patients with preserved LV function. In individuals with concomitant LV dysfunction and cAF, APD was determined by LV dysfunction-associated prolongation and by cAF-dependent shortening, respectively, consistent with changes in K2P3.1 abundance. K2P3.1 inhibition attenuated APD shortening in cAF patients irrespective of LV function, whereas in pAF subjects with severely reduced LV function, K2P3.1 blockade resulted in disproportionately high APD prolongation. CONCLUSION: LV dysfunction is associated with reduction of atrial K2P3.1 channel expression, while cAF leads to increased K2P3.1 abundance. Differential remodelling of K2P3.1 and APD provides a basis for patient-tailored antiarrhythmic strategies.

Slow Activity in Focal Epilepsy During Sleep and Wakefulness.

INTRODUCTION: We aimed to test differences between healthy subjects and patients with respect to slow wave activity during wakefulness and sleep. METHODS: Fifteen patients affected by nonlesional focal epilepsy originating within temporal areas and fourteen matched controls underwent a 24-hour EEG recording. We studied the EEG power spectral density during wakefulness and sleep in delta (1-4 Hz), theta (5-7 Hz), alpha (8-11 Hz), sigma (12-15 Hz), and beta (16-20 Hz) bands. RESULTS: During sleep, patients with focal epilepsy showed higher power from delta to beta frequency bands compared with controls. The effect was widespread for alpha band and above, while localized over the affected hemisphere for delta (sleep cycle 1, P = .006; sleep cycle 2, P = .008; sleep cycle 3, P = .017). The analysis of interhemispheric differences showed that the only frequency band stronger over the affected regions was the delta band during the first 2 sleep cycles (sleep cycle 1, P = .014; sleep cycle 2, P = .002). During wakefulness, patients showed higher delta/theta activity over the affected regions compared with controls. CONCLUSIONS: Patients with focal epilepsy showed a pattern of power increases characterized by a selective slow wave activity enhancement over the epileptic regions during daytime and sleep. This phenomenon was stronger and asymmetric during the first sleep cycles.

A novel c132-134del mutation in Unverricht-Lundborg disease and the review of literature of heterozygous compound patients.

Unverricht-Lundborg disease or progressive myoclonic epilepsy type 1 (EPM1) is an autosomal recessive disease caused by mutation of the cystatin B gene (CSTB), located on chromosome 21q22.3. The most common mutation is an expansion of unstable dodecamer repetition (CCCCGCCCCGCG), whereas other types of mutations are rare. Among these, heterozygous compound mutations are described to induce a more severe phenotype than that of homozygous dodecameric repetition. We report two siblings affected by heterozygous compound mutations carrying a novel mutation of the deletion of three nucleotides in exon 2 of the gene in position 132-134 of the coding sequence (c.132-134del) in the allele not including the dodecamer repetition. This mutation results in the loss of two amino acid residues and insertion of an asparagine in position 44 (p.Lys44_Ser45delinsAsn). Our patients presented a very different clinical picture. The male patient had a severe myoclonus, drug-resistant epilepsy and psychiatric comorbidity, while his affected sister had only very rare seizures and sporadic myoclonic jerks at awakening. The revision of literature about heterozygous compound EPM1 patients confirms this gender phenotypic expressivity, with female patients carrying less severe symptoms than male patients. These data lead to the hypothesis of complex gender-specific factors interacting with CSTB expressivity in EPM1 patients.

Hyperventilation induces sympathetic overactivation in mesial temporal epilepsy.

OBJECTIVE: Hyperventilation (HV) during electroencephalography (EEG) is a standard clinical procedure to trigger seizures in patients affected by mesial temporal lobe epilepsy (MTLE). Despite the pathophysiology of this susceptibility to HV is not definitively understood, it may be hypothesized to be related to ictal and interictal sympathetic nervous system abnormalities, the presence of which is well known in MTLE patients. In order to test this hypothesis we investigated the HV effect on heart rate variability (HRV) in a group of MTLE patients, compared to a matched group of healthy controls. MATERIAL AND METHODS: Forty patients affected by MTLE and 40 age- and sex-matched controls were enrolled in the study. In those subjects, a standard electroencephalographic recording has been acquired and the high and the low frequency components (HF, LF) of heart rate variability (HRV) and their ratio (LF/HF) have been analyzed at rest and during the HV. Indeed, LF/HF is a reliable index of sympathetic tone modulation. RESULTS: HRV did not differ between MTLE and healthy subjects at rest, whereas HV induced a significant LF/HF increase only in MTLE. Within the MTLE group, males showed higher LF/HF increase during HV respect to females, while no differences related to the side of the epileptic focus were found. DISCUSSION: MTLE patients showed an increased sympathetic response to HV compared to healthy subjects. HRV analysis points towards an autonomic overactivation as a pathophysiological pathway subtending seizure triggered by hyperventilation in MTLE. Autonomic susceptibility in MTLE may help to explain the increased prevalence of arrhythmic events in these patients, potentially predisposing to Sudden Unexpected Death in Epilepsy (SUDEP).