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Data concerning the combined prognostic role of natriuretic peptide (NP) and troponin in patients with COVID-19 are lacking. The aim of the study is to evaluate the combined prognostic value of NPs and troponin in hospitalized COVID-19 patients. From March 1, 2020 to April 9, 2020, consecutive patients with COVID-19 and available data on cardiac biomarkers at admission were recruited. Patients admitted for acute coronary syndrome were excluded. Troponin levels were defined as elevated when greater than the 99th percentile of normal values. NPs were considered elevated if above the limit for ruling in acute heart failure (HF). A total of 341 patients were included in this study, mean age 68 ± 13 years, 72% were men. During a median follow-up period of 14 days, 81 patients (24%) died. In the Cox regression analysis, patients with elevated both NPs and troponin levels had higher risk of death compared with those with normal levels of both (hazard ratio 2.94; 95% confidence interval 1.31 to 6.64; p = 0.009), and this remained significant after adjustment for age, gender, oxygen saturation, HF history, and chronic kidney disease. Interestingly, NPs provided risk stratification also in patients with normal troponin values (hazard ratio 2.86; 95% confidence interval 1.21 to 6.72; p = 0.016 with high NPs levels). These data show the combined prognostic role of troponin and NPs in COVID-19 patients. NPs value may be helpful in identifying patients with a worse prognosis among those with normal troponin values. Further, NPs’ cut-point used for diagnosis of acute HF has a predictive role in patients with COVID-19.

IntroductionThe role of sex compared to comorbidities and other prognostic variables in patients with coronavirus disease (COVID-19) is unclear.MethodsThis is a retrospective observational study on patients with COVID-19 infection, referred to 13 cardiology units. The primary objective was to assess the difference in risk of death between the sexes. The secondary objective was to explore sex-based heterogeneity in the association between demographic, clinical and laboratory variables, and patients' risk of death.ResultsSeven hundred and one patients were included: 214 (30.5%) women and 487 (69.5%) men. During a median follow-up of 15-days, deaths occurred in 39 (18.2%) women and 126 (25.9%) men. In a multivariable Cox regression model, men had a nonsignificantly higher risk of death vs. women (P = 0.07).The risk of death was more than double in men with a low lymphocytes count as compared with men with a high lymphocytes count [overall survival hazard ratio (OS-HR) 2.56, 95% confidence interval (CI) 1.72-3.81]. In contrast, lymphocytes count was not related to death in women (P=0.03).Platelets count was associated with better outcome in men (OS-HR for increase of 50-×-103units: 0.88 95% CI 0.78-1.00) but not in women. The strength of association between higher PaO2/FiO2ratio and lower risk of death was larger in women (OS-HR for increase of 50-mmHg/%: 0.72, 95% CI 0.59-0.89) vs. men (OS-HR: 0.88, 95% CI 0.80-0.98; P-=-0.05).ConclusionsPatients' sex is a relevant variable that should be taken into account when evaluating risk of death from COVID-19. There is a sex-based heterogeneity in the association between baseline variables and patients' risk of death.

AIMS: Current long QT syndrome (LQTS) therapy, largely based on beta-blockade, does not prevent arrhythmias in all patients; therefore, novel therapies are warranted. Pharmacological inhibition of the serum/glucocorticoid-regulated kinase 1 (SGK1-Inh) has been shown to shorten action potential duration (APD) in LQTS type 3. We aimed to investigate whether SGK1-Inh could similarly shorten APD in LQTS types 1 and 2. METHODS AND RESULTS: Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and hiPSC-cardiac cell sheets (CCS) were obtained from LQT1 and LQT2 patients; CMs were isolated from transgenic LQT1, LQT2, and wild-type (WT) rabbits. Serum/glucocorticoid-regulated kinase 1 inhibition effects (300 nM-10 µM) on field potential durations (FPD) were investigated in hiPSC-CMs with multielectrode arrays; optical mapping was performed in LQT2 CCS. Whole-cell and perforated patch clamp recordings were performed in isolated LQT1, LQT2, and WT rabbit CMs to investigate SGK1-Inh (3 µM) effects on APD. In all LQT2 models across different species (hiPSC-CMs, hiPSC-CCS, and rabbit CMs) and independent of the disease-causing variant (KCNH2-p.A561V/p.A614V/p.G628S/IVS9-28A/G), SGK1-Inh dose-dependently shortened FPD/APD at 0.3-10 µM (by 20-32%/25-30%/44-45%). Importantly, in LQT2 rabbit CMs, 3 µM SGK1-Inh normalized APD to its WT value. A significant FPD shortening was observed in KCNQ1-p.R594Q hiPSC-CMs at 1/3/10 µM (by 19/26/35%) and in KCNQ1-p.A341V hiPSC-CMs at 10 µM (by 29%). No SGK1-Inh-induced FPD/APD shortening effect was observed in LQT1 KCNQ1-p.A341V hiPSC-CMs or KCNQ1-p.Y315S rabbit CMs at 0.3-3 µM. CONCLUSION: A robust SGK1-Inh-induced APD shortening was observed across different LQT2 models, species, and genetic variants but less consistently in LQT1 models. This suggests a genotype- and variant-specific beneficial effect of this novel therapeutic approach in LQTS.

Background: The proliferation of genetic profiling has revealed many associations between genetic variations and disease. However, large-scale phenotyping efforts in largely healthy populations, coupled with DNA sequencing, suggest variants currently annotated as pathogenic are more common in healthy populations than previously thought. In addition, novel and rare variants are frequently observed in genes associated with disease both in healthy individuals and those under suspicion of disease. This raises the question of whether these variants can be useful predictors of disease. To answer this question, we assessed the degree to which the presence of a variant in the cardiac potassium channel gene KCNH2 was diagnostically predictive for the autosomal dominant long QT syndrome. Methods: We estimated the probability of a long QT diagnosis given the presence of each KCNH2 variant using Bayesian methods that incorporated variant features such as changes in variant function, protein structure, and in silico predictions. We call this estimate the posttest probability of disease. Our method was applied to over 4000 individuals heterozygous for 871 missense or in-frame insertion/deletion variants in KCNH2 and validated against a separate international cohort of 933 individuals heterozygous for 266 missense or in-frame insertion/deletion variants. Results: Our method was well-calibrated for the observed fraction of heterozygotes diagnosed with long QT syndrome. Heuristically, we found that the innate diagnostic information one learns about a variant from 3-dimensional variant location, in vitro functional data, and in silico predictors is equivalent to the diagnostic information one learns about that same variant by clinically phenotyping 10 heterozygotes. Most importantly, these data can be obtained in the absence of any clinical observations. Conclusions: We show how variant-specific features can inform a prior probability of disease for rare variants even in the absence

Precision Medicine (PM) is an innovative approach that, by relying on large populations' datasets, patients' genetics and characteristics, and advanced technologies, aims at improving risk stratification and at identifying patient-specific management through targeted diagnostic and therapeutic strategies. Cardiac channelopathies are being progressively involved in the evolution brought by PM and some of them are benefiting from these novel approaches, especially the long QT syndrome. Here, we have explored the main layers that should be considered when developing a PM approach for cardiac channelopathies, with a focus on modern in vitro strategies based on patient-specific human-induced pluripotent stem cells and on in silico models. PM is where scientists and clinicians must meet and integrate their expertise to improve medical care in an innovative way but without losing common sense. We have indeed tried to provide the cardiologist's point of view by comparing state-of-the-art techniques and approaches, including revolutionary discoveries, to current practice. This point matters because the new approaches may, or may not, exceed the efficacy and safety of established therapies. Thus, our own eagerness to implement the most recent translational strategies for cardiac channelopathies must be tempered by an objective assessment to verify whether the PM approaches are indeed making a difference for the patients. We believe that PM may shape the diagnosis and treatment of cardiac channelopathies for years to come. Nonetheless, its potential superiority over standard therapies should be constantly monitored and assessed before translating intellectually rewarding new discoveries into clinical practice.

Aims: To assess the clinical relevance of a history of atrial fibrillation (AF) in hospitalized patients with coronavirus disease 2019 (COVID-19). Methods and results: We enrolled 696 consecutive patients (mean age 67.4 ± 13.2 years, 69.7% males) admitted for COVID-19 in 13 Italian cardiology centres between 1 March and 9 April 2020. One hundred and six patients (15%) had a history of AF and the median hospitalization length was 14 days (interquartile range 9-24). Patients with a history of AF were older and with a higher burden of cardiovascular risk factors. Compared to patients without AF, they showed a higher rate of in-hospital death (38.7% vs. 20.8%; P < 0.001). History of AF was associated with an increased risk of death after adjustment for clinical confounders related to COVID-19 severity and cardiovascular comorbidities, including history of heart failure (HF) and increased plasma troponin [adjusted hazard ratio (HR): 1.73; 95% confidence interval (CI) 1.06-2.84; P = 0.029]. Patients with a history of AF also had more in-hospital clinical events including new-onset AF (36.8% vs. 7.9%; P < 0.001), acute HF (25.3% vs. 6.3%; P < 0.001), and multiorgan failure (13.9% vs. 5.8%; P = 0.010). The association between AF and worse outcome was not modified by previous or concomitant use of anticoagulants or steroid therapy (P for interaction >0.05 for both) and was not related to stroke or bleeding events. Conclusion: Among hospitalized patients with COVID-19, a history of AF contributes to worse clinical course with a higher mortality and in-hospital events including new-onset AF, acute HF, and multiorgan failure. The mortality risk remains significant after adjustment for variables associated with COVID-19 severity and comorbidities.

Background: Glucocorticoid therapy has emerged as an effective therapeutic option in hospitalized patients with coronavirus disease 2019 (COVID-19). This study aimed to focus on the impact of relevant clinical and laboratory factors on the protective effect of glucocorticoids on mortality. Methods: A sub-analysis was performed of the multicenter Cardio-COVID-Italy registry, enrolling consecutive patients with COVID-19 admitted to 13 Italian cardiology units between 01 March 2020 and 09 April 2020. The primary endpoint was in-hospital mortality. Results: A total of 706 COVID-19 patients were included (349 treated with glucocorticoids, 357 not treated with glucocorticoids). After adjustment for relevant covariates, use of glucocorticoids was associated with a lower risk of in-hospital mortality (adjusted HR 0.44; 95% CI 0.26–0.72; p = 0.001). A significant interaction was observed between the protective effect of glucocorticoids on mortality and PaO2/FiO2 ratio on admission (p = 0.042), oxygen saturation on admission (p = 0.017), and peak CRP (0.023). Such protective effects of glucocorticoids were mainly observed in patients with lower PaO2/FiO2 ratio (<300), lower oxygen saturation ([removed]100 mg/L). Conclusions: The protective effects of glucocorticoids on mortality in COVID-19 were more evident among patients with worse respiratory parameters and higher systemic inflammation.

Aims: Myocardial injury (MI) in coronavirus disease-19 (COVID-19) is quite prevalent at admission and affects prognosis. Little is known about troponin trajectories and their prognostic role. We aimed to describe the early in-hospital evolution of MI and its prognostic impact. Methods and results: We performed an analysis from an Italian multicentre study enrolling COVID-19 patients, hospitalized from 1 March to 9 April 2020. MI was defined as increased troponin level. The first troponin was tested within 24 h from admission, the second one between 24 and 48 h. Elevated troponin was defined as values above the 99th percentile of normal values. Patients were divided in four groups: normal, normal then elevated, elevated then normal, and elevated. The outcome was in-hospital death. The study population included 197 patients; 41% had normal troponin at both evaluations, 44% had elevated troponin at both assessments, 8% had normal then elevated troponin, and 7% had elevated then normal troponin. During hospitalization, 49 (25%) patients died. Patients with incident MI, with persistent MI, and with MI only at admission had a higher risk of death compared with those with normal troponin at both evaluations (P < 0.001). At multivariable analysis, patients with normal troponin at admission and MI injury on Day 2 had the highest mortality risk (hazard ratio 3.78, 95% confidence interval 1.10–13.09, P = 0.035). Conclusions: In patients admitted for COVID-19, re-test MI on Day 2 provides a prognostic value. A non-negligible proportion of patients with incident MI on Day 2 is identified at high risk of death only by the second measurement.

Background: Pulmonary embolism (PE) has been described in coronavirus disease 2019 (COVID-19) critically ill patients, but the evidence from more heterogeneous cohorts is limited. Methods: Data were retrospectively obtained from consecutive COVID-19 patients admitted to 13 Cardiology Units in Italy, from March 1st to April 9th, 2020, and followed until in-hospital death, discharge, or April 23rd, 2020. The association of baseline variables with computed tomography-confirmed PE was investigated by Cox hazards regression analysis. The relationship between d-dimer levels and PE incidence was evaluated using restricted cubic splines models. Results: The study included 689 patients (67.3 ± 13.2 year-old, 69.4% males), of whom 43.6% were non-invasively ventilated and 15.8% invasively. 52 (7.5%) had PE over 15 (9–24) days of follow-up. Compared with those without PE, these subjects had younger age, higher BMI, less often heart failure and chronic kidney disease, more severe cardio-pulmonary involvement, and higher admission d-dimer [4344 (1099–15,118) vs. 818.5 (417–1460) ng/mL, p < 0.001]. They also received more frequently darunavir/ritonavir, tocilizumab and ventilation support. Furthermore, they faced more bleeding episodes requiring transfusion (15.6% vs. 5.1%, p < 0.001) and non-significantly higher in-hospital mortality (34.6% vs. 22.9%, p = 0.06). In multivariate regression, only d-dimer was associated with PE (HR 1.72, 95% CI 1.13–2.62; p = 0.01). The relation between d-dimer concentrations and PE incidence was linear, without inflection point. Only two subjects had a baseline d-dimer < 500 ng/mL. Conclusions: PE occurs in a sizable proportion of hospitalized COVID-19 patients. The implications of bleeding events and the role of d-dimer in this population need to be clarified. Graphic abstract: [Figure not available: see fulltext.].

We generated human induced pluripotent stem cells (hiPSCs) from dermal fibroblasts of a 40 years old female patient homozygous for the mutation c.535 G > A p.G179S on the KCNQ1 gene, causing a severe form of autosomal recessive Long QT Syndrome type 1 (AR-LQT1). The hiPSCs, generated using classical approach of the four retroviruses enconding the reprogramming factors OCT4, SOX2, cMYC and KLF4, display pluripotent stem cell characteristics, and differentiate into cell lineages of all three germ layers: endoderm, mesoderm and ectoderm.

Background: Few studies prospectively assessed risk factors for ventricular fibrillation (VF) during a first myocardial infarction (MI). We designed a nation-wide study aiming to identify clinical and genetic characteristics associated with primary VF; and report here about clinical features. Methods: PREDESTINATION (PRimary vEntricular fibrillation and suDden dEath during a firST myocardIal iNfArcTION) is an Italian case-control, prospective multicentre study. Cases are patients aged 18–80 years with a first MI and at least one VF episodes occurring within 24 h of symptoms onset, before reperfusion. Cases and controls are paired 1: 2 by gender and age (±5 years). Results: Among 1026 patients enrolled between 2007 and 2017, 970 entered the primary analysis: 375 cases and 595 controls (mean age 59 years, 85% males). Multivariable analysis identified 5 independent predictors of primary VF: systolic blood pressure (OR 0.982, 95% CI: 0.98–0.99 for each mm Hg) and K+ levels <3.5 mEq/L at presentation (OR 2.28, 95% CI: 1.6–3.3), family history of sudden death (OR 1.80, 95% CI: 1.1–3.0), physical inactivity (OR 1.73, 95% CI: 1.1–2.8) and anterior MI (OR 1.52, 95% CI: 1.1–2.1). Excluding K+ levels obtained after VF, the OR associated with K+ levels <3.5 mEq/L was1.99 (95 CI 1.22–3.21). Conclusions: The present study identified 5 independent predictors of primary VF: familiarity, anterior MI, low systolic blood pressure, physical inactivity and hypokalaemia. Importantly, the last two risk factors are modifiable and, especially in the presence of a family history of sudden death, they should be avoided as much as possible.

Importance: Myocardial injury, detected by elevated plasma troponin levels, has been associated with mortality in patients hospitalized with coronavirus disease 2019 (COVID-19). However, the initial data were reported from single-center or 2-center studies in Chinese populations. Compared with these patients, European and US patients are older, with more comorbidities and higher mortality rates. Objective: To evaluate the prevalence and prognostic value of myocardial injury, detected by elevated plasma troponin levels, in a large population of White Italian patients with COVID-19. Design, Setting, and Participants: This is a multicenter, cross-sectional study enrolling consecutive patients with laboratory-confirmed COVID-19 who were hospitalized in 13 Italian cardiology units from March 1 to April 9, 2020. Patients admitted for acute coronary syndrome were excluded. Elevated troponin levels were defined as values greater than the 99th percentile of normal values. Main Outcomes and Measures: Clinical characteristics and outcomes stratified as elevated or normal cardiac troponin levels at admission, defined as troponin T or troponin I at a level greater than the 99th percentile of normal values. Results: A total of 614 patients with COVID-19 were included in this study (mean age [SD], 67 [13] years; 70.8% male), of whom 148 patients (24.1%) died during the hospitalization. Elevated troponin levels were found in 278 patients (45.3%). These patients were older (mean [SD] age, 64.0 [13.6] years vs 71.3 [12.0] years; P <.001) and had higher prevalence of hypertension (168 patients [50.5%] vs 182 patients [65.9%]; P <.001), heart failure (24 [7.2%]; 63 [22.8%]; P <.001), coronary artery disease (50 [15.0%] vs 87 [31.5%]; P <.001), and atrial fibrillation (33 [9.9%] vs 67 [24.3%]; P <.001). Elevated troponin levels were associated with an increased in-hospital mortality (37% vs 13%; HR, 1.71 [95% CI, 1.13-2.59]; P =.01 via multivariable Cox regression analysi

Aims: To evaluate the impact of the COVID-19 pandemic on patient admissions to Italian cardiac care units (CCUs). Methods and Results: We conducted a multicentre, observational, nationwide survey to collect data on admissions for acute myocardial infarction (AMI) at Italian CCUs throughout a 1 week period during the COVID-19 outbreak, compared with the equivalent week in 2019. We observed a 48.4% reduction in admissions for AMI compared with the equivalent week in 2019 (P < 0.001). The reduction was significant for both ST-segment elevation myocardial infarction [STEMI; 26.5%, 95% confidence interval (CI) 21.7-32.3; P = 0.009] and non-STEMI (NSTEMI; 65.1%, 95% CI 60.3-70.3; P < 0.001). Among STEMIs, the reduction was higher for women (41.2%; P = 0.011) than men (17.8%; P = 0.191). A similar reduction in AMI admissions was registered in North Italy (52.1%), Central Italy (59.3%), and South Italy (52.1%). The STEMI case fatality rate during the pandemic was substantially increased compared with 2019 [risk ratio (RR) = 3.3, 95% CI 1.7-6.6; P < 0.001]. A parallel increase in complications was also registered (RR = 1.8, 95% CI 1.1-2.8; P = 0.009). Conclusion: Admissions for AMI were significantly reduced during the COVID-19 pandemic across Italy, with a parallel increase in fatality and complication rates. This constitutes a serious social issue, demanding attention by the scientific and healthcare communities and public regulatory agencies.

Aims: To assess the prognostic value of a history of heart failure (HF) in patients with coronavirus disease 2019 (COVID-19). Methods and results: We enrolled 692 consecutive patients admitted for COVID-19 in 13 Italian cardiology centres between 1 March and 9 April 2020. Mean age was 67.4 ± 13.2 years, 69.5% of patients were males, 90 (13.0%) had a history of HF, median hospitalization length was 14 days (interquartile range 9–24). In-hospital death occurred in 37 of 90 patients (41.1%) with HF history vs. 126 of those with no HF history (20.9%). The increased risk of death associated with HF history remained significant after adjustment for clinical variables related to COVID-19 and HF severity, including comorbidities, oxygen saturation, lymphocyte count and plasma troponin [adjusted hazard ratio (HR) for death: 2.25; 95% confidence interval (CI) 1.26–4.02; P = 0.006 at multivariable Cox regression model including 404 patients]. Patients with a history of HF also had more in-hospital complications including. acute HF (33.3% vs. 5.1%, P < 0.001), acute renal failure (28.1% vs. 12.9%, P < 0.001), multiorgan failure (15.9% vs. 5.8%, P = 0.004) and sepsis (18.4% vs. 8.9%, P = 0.006). Other independent predictors of outcome were age, sex, oxygen saturation and oxygen partial pressure at arterial gas analysis/fraction of inspired oxygen ratio (PaO2/FiO2). In-hospital treatment with corticosteroids and heparin had beneficial effects (adjusted HR for death: 0.46; 95% CI 0.29–0.74; P = 0.001; n = 404 for corticosteroids, and adjusted HR 0.41; 95% CI 0.25–0.67; P < 0.001; n = 364 for heparin). Conclusions: Hospitalized patients with COVID-19 and a history of HF have an extremely poor outcome with higher mortality and in-hospital complications. HF history is an independent predictor of increased in-hospital mortality.

In the early phases of the COVID-19 pandemic, drug repurposing was widely used to identify compounds that could improve the prognosis of symptomatic patients infected by SARS-CoV-2. Hydroxychloroquine (HCQ) was one of the first drugs used to treat COVID-19 due to its supposed capacity of inhibiting SARS-CoV-2 infection and replication in vitro. While its efficacy is debated, HCQ has been associated with QT interval prolongation and potentially Torsades de Pointes, especially in patients predisposed to developing drug-induced Long QT Syndrome (LQTS) as silent carriers of variants associated with congenital LQTS. If confirmed, these effects represent a limitation to the at-home use of HCQ for COVID-19 infection as adequate ECG monitoring is challenging. We investigated the proarrhythmic profile of HCQ with Multi-Electrode Arrays after exposure of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from two healthy donors, one asymptomatic and two symptomatic LQTS patients. We demonstrated that: I) HCQ induced a concentration-dependent Field Potential Duration (FPD) prolongation and halted the beating at high concentration due to the combined effect of HCQ on multiple ion currents. II) hiPSC-CMs from healthy or asymptomatic carriers tolerated higher concentrations of HCQ and showed lower susceptibility to HCQ-induced electrical abnormalities regardless of baseline FPD. These findings agree with the clinical safety records of HCQ and demonstrated that hiPSC-CMs potentially discriminates symptomatic vs. asymptomatic mutation carriers through pharmacological interventions. Disease-specific cohorts of hiPSC-CMs may be a valid preliminary addition to assess drug safety in vulnerable populations, offering rapid preclinical results with valuable translational relevance for precision medicine.

We generated human induced pluripotent stem cells (hiPSCs) from dermal fibroblasts of a male carrier of the heterozygous mutation c.1781 G > A p.R594Q on the KCNQ1 gene. hiPSCs, generated using four retroviruses each encoding for OCT4, SOX2, KLF4 and cMYC, display pluripotent stem cell characteristics, and can be differentiated into spontaneously beating cardiomyocytes (hiPSC-CMs).

We generated human induced pluripotent stem cells (hiPSCs) from a symptomatic Long QT Syndrome (LQTS) type 1 patient, belonging to a South African (SA) founder population segregating the heterozygous mutation c.1022C > T p.A341V on the KCNQ1 gene. The patient is also homozygous for the two minor variants rs4657139 and rs16847548 on the NOS1AP gene, associated with greater risk for cardiac arrest and sudden death in LQTS mutation carriers of the founder population. hiPSCs, obtained using four retroviruses encoding the reprogramming factors OCT4, SOX2, cMYC and KLF4, display pluripotent stem cell characteristics, and can be differentiated into spontaneously beating cardiomyocytes (hiPSC-CMs).

We generated human induced pluripotent stem cells (hiPSCs) from dermal fibroblasts of a woman carrier of the heterozygous mutation c.568C > T p.R190W on the KCNQ1 gene. hiPSCs, obtained using four retroviruses enconding the reprogramming factors OCT4, SOX2, cMYC and KLF4, display pluripotent stem cell characteristics, and can be differentiated into spontaneously beating cardiomyocytes (hiPSC-CMs).

We generated PSMi001-A and PSMi008-A hiPSC lines from two individuals belonging to a South African (SA) founder population in which the malignant KCNQ1-A341V mutation cosegregates with the Long QT Syndrome (LQTS) phenotype. PSMi001-A was derived from an asymptomatic KCNQ1-A341V mutation carrier, whereas PSMi008-A was derived from a healthy non-mutation carrier, heterozygous for the minor variant rs16847548 on the NOS1AP gene, associated with QT prolongation in the general population, and with a greater risk for cardiac arrest in the affected members of the SA founder population. The hiPSCs, generated using the Yamanaka's retroviruses, display pluripotent stem cell features and trilineage differentiation potential.

The discovery of a genetic basis for cardiac repolarization disorders has introduced innovative technologies and concepts in the field of cardiac arrhythmias and has revolutionized the knowledge of these disorders as well as patients’ treatment. Conventional methodologies for the in vitro study of cardiac arrhythmias, only indirectly linked to the clinical phenotype, have started to age, and the information they can now provide suddenly appears limited. After the discovery that patient-specific cardiomyocytes can be derived, in virtually unlimited numbers, from pluripotent stem cells, we are now on the edge of another breakthrough with basic science laboratories heavily linked to clinical practice and offering tools with substantially higher translational capabilities. In this chapter, we present an excursus on the path that has led to the discovery, optimization, and implementation of pluripotent stem cell-derived cardiomyocytes for cardiac disease modeling. Then, we cover the major repolarization disorders with genetic bases whose phenotypes have been recapitulated and studied with these cardiomyocytes. Along this, we describe the techniques currently used to study repolarization disorders in vitro, and we offer a glimpse on what will come next in this field. Finally, we analyze the translational relevance of the powerful combination between genetics and stem cell-based approaches for cardiac arrhythmias, in a context of precision medicine.

Long QT syndrome (LQTS) is a potentially severe arrhythmogenic disorder, associated with a prolonged QT interval and sudden death, caused by mutations in key genes regulating cardiac electrophysiology. Current strategies to study LQTS in vitro include heterologous systems or animal models. Despite their value, the overwhelming power of genetic tools has exposed the many limitations of these technologies. In 2010, human-induced pluripotent stem cells (hiPSCs) revolutionised the field and allowed scientists to study in vitro some of the disease traits of LQTS on hiPSC-derived cardiomyocytes (hiPSC-CMs) from LQTS patients. In this concise review we present how the hiPSC technology has been used to model three main forms of LQTS and the severe form of LQTS associated with mutations in calmodulin. We also introduce some of the most recent challenges that must be tackled in the upcoming years to successfully shift hiPSC-CMs from powerful in vitro disease modelling tools into assets to improve risk stratification and clinical decision-making.

Aims: In long QT syndrome (LQTS) patients, modifier genes modulate the arrhythmic risk associated with a disease-causing mutation. Their recognition can improve risk stratification and clinical management, but their discovery represents a challenge. We tested whether a cellular-driven approach could help to identify new modifier genes and especially their mechanism of action. Methods and results: We generated human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) from two patients carrying the same KCNQ1-Y111C mutation, but presenting opposite clinical phenotypes. We showed that the phenotype of the iPSC-CMs derived from the symptomatic patient is due to impaired trafficking and increased degradation of the mutant KCNQ1 and wild-type human ether-a-go-go-related gene. In the iPSC-CMs of the asymptomatic (AS) patient, the activity of an E3 ubiquitin-protein ligase (Nedd4L) involved in channel protein degradation was reduced and resulted in a decreased arrhythmogenic substrate. Two single-nucleotide variants (SNVs) on the Myotubularin-related protein 4 (MTMR4) gene, an interactor of Nedd4L, were identified by whole-exome sequencing as potential contributors to decreased Nedd4L activity. Correction of these SNVs by CRISPR/Cas9 unmasked the LQTS phenotype in AS cells. Importantly, the same MTMR4 variants were present in 77% of AS Y111C mutation carriers of a separate cohort. Thus, genetically mediated interference with Nedd4L activation seems associated with protective effects. Conclusion: Our finding represents the first demonstration of the cellular mechanism of action of a protective modifier gene in LQTS. It provides new clues for advanced risk stratification and paves the way for the design of new therapies targeting this specific molecular pathway.

We report the generation of human induced pluripotent stem cells (hiPSCs) from dermal fibroblasts of a female patient carrier of the two compound heterozygous mutations c.568 C>T p.R190W (maternal allele), and c.1781 G>A p.R594Q (paternal allele) on the KCNQ1 gene, causing Jervell and Lange-Nielsen Syndrome (JLNS). To obtain hiPSCs, we used the classical approach of the four retroviruses each encoding for a reprogramming factor OCT4, SOX2, KLF4, cMYC. The obtained hiPSC clones display pluripotent stem cell characteristics, and differentiate into spontaneously beating cardiomyocytes (hiPSC-CMs).

We generated human induced pluripotent stem cells (hiPSCs) from dermal fibroblasts of a 51 years old female patient homozygous for the mutation c.535 G>A p.G179S on the KCNQ1 gene, causing a severe form of autosomal recessive Long QT Syndrome type 1 (AR-LQT1), not associated with deafness. The hiPSCs, generated using four retroviruses each encoding for a reprogramming factor OCT4, SOX2, KLF4, cMYC, are pluripotent and can differentiate into spontaneously beating cardiomyocytes (hiPSC-CMs).

Aims: To evaluate the impact of the COVID-19 pandemic on patient admissions to Italian cardiac care units (CCUs). Methods and Results: We conducted a multicentre, observational, nationwide survey to collect data on admissions for acute myocardial infarction (AMI) at Italian CCUs throughout a 1 week period during the COVID-19 outbreak, compared with the equivalent week in 2019. We observed a 48.4% reduction in admissions for AMI compared with the equivalent week in 2019 (P < 0.001). The reduction was significant for both ST-segment elevation myocardial infarction [STEMI; 26.5%, 95% confidence interval (CI) 21.7-32.3; P = 0.009] and non-STEMI (NSTEMI; 65.1%, 95% CI 60.3-70.3; P < 0.001). Among STEMIs, the reduction was higher for women (41.2%; P = 0.011) than men (17.8%; P = 0.191). A similar reduction in AMI admissions was registered in North Italy (52.1%), Central Italy (59.3%), and South Italy (52.1%). The STEMI case fatality rate during the pandemic was substantially increased compared with 2019 [risk ratio (RR) = 3.3, 95% CI 1.7-6.6; P < 0.001]. A parallel increase in complications was also registered (RR = 1.8, 95% CI 1.1-2.8; P = 0.009). Conclusion: Admissions for AMI were significantly reduced during the COVID-19 pandemic across Italy, with a parallel increase in fatality and complication rates. This constitutes a serious social issue, demanding attention by the scientific and healthcare communities and public regulatory agencies.

Aims Having shown that Lumacaftor rescued the hERG trafficking defect in the induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) of two LQT2 patients, we tested whether the commercial association Lumacaftor þ Ivacaftor (LUM þ IVA) could shorten the QTc in the same two patients. Methods After hospital admission and 1 day of baseline recordings, half dose LUM þ IVA was administered on Day 1, fol- and results lowed by full dose (LUM 800 mg þ IVA 500 mg) for 7 days. A continuous 12-lead Holter ECG allowed a large number of blind QTc measurements. Lumacaftor þ Ivacaftor shortened QTc significantly in both patients: in V6 from 551 ± 22 ms to 523 ± 35 ms in Patient 1 (Pt1) and from 472 ± 21 ms to 449 ± 20 ms in Patient 2 (Pt2); in DII from 562 ± 25 ms to 549 ± 35 ms in Pt1 and from 485 ± 32 ms to 452 ± 18 ms in Pt2. In both patients, the percentage of QTc values in the lower tertile increased strikingly: in V6 from 33% to 68% and from 33% to 76%; in DII from 33% to 50% and from 33% to 87%. In the wash-out period a rebound in QTc was observed. On treatment, both patients developed diarrhoea, Pt1 more than Pt2. Conclusion This represents the first attempt to validate in patients the in vitro results of a drug repurposing strategy for cardiovascular disorders. Lumacaftor þ Ivacaftor shortened significantly the QTc in the two LQT2 patients with a trafficking defect, largely confirming the findings in their iPSC-CMs but with smaller quantitative changes. The findings are encouraging but immediate translation into clinical practice, without validation in more patients, would be premature.

Several studies have demonstrated that miRNA are involved in cardiac development, stem cell maintenance, and differentiation. In particular, it has been shown that miRNA133, miRNA1, and miRNA499 are involved in progenitor cell differentiation into cardiomyocytes. However, it is unknown whether different miRNA may act synergistically to improve cardiac differentiation. We used mouse P19 cells as a cardiogenic differentiation model. miRNA499, miRNA1, or miRNA133 were transiently over-expressed in P19 cells individually or in different combinations. The over-expression of miRNA499 alone increased the number of beating cells and the association of miRNA499 with miRNA133 exerted a synergistic effect, further increasing the number of beating cells. Real-time polymerase chain reaction showed that the combination of miRNA499-‰+-‰133 enhanced the expression of cardiac genes compared with controls. Western blot and immunocytochemistry for connexin43 and cardiac troponin T confirmed these findings. Importantly, caffeine responsiveness, a clear functional parameter of cardiac differentiation, was increased by miRNA499 in association with miRNA133 and was directly correlated with the activation of the cardiac troponin I isoform promoter. Cyclic contractions were reversibly abolished by extracellular calcium depletion, nifedipine, ryanodine, and IP3R blockade. Finally, we demonstrated that the use of miRNA499-‰+-‰133 induced cardiac differentiation even in the absence of dimethyl sulfoxide. Our results show that the areas spontaneously contracting possess electrophysiological and pharmacological characteristics compatible with true cardiac excitation-contraction coupling. The translational relevance of our findings was reinforced by the demonstration that the over-expression of miRNA499 and miRNA133 was also able to induce the differentiation of human mesenchymal stromal cells toward the cardiac lineage.