A gain-of-function HCN4 mutant in the HCN domain is responsible for inappropriate sinus tachycardia in a Spanish family.

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.

Intermuscular S-ICD Implantation in Pediatric Patients.

The use of conventional implantable cardioverter-defibrillators (ICDs) in children presents important technical challenges. We present the surgical technique necessary to adapt the subcutaneous ICD (S-ICD) implantation designed for adults, to children, including patients weighing less than 20 kg. The implant procedure implies a two-incision technique and interfascial serratus anterior-latissimus dorsi dissection to accommodate the device. S-ICD implantation was successfully performed in three patients of 19, 28, and 24 kg, respectively, two of them suffered cardiorespiratory arrest. Intermuscular thoracic implantation of S-ICD might represent an effective strategy for primary or secondary prevention of sudden cardiac death in pediatric patients.

Modeling a Dynamic Printability Window on Polysaccharide Blend Inks for Extrusion Bioprinting.

Extrusion-based bioprinting is one of the most widespread technologies due to its affordability, wide range of processable materials, and ease of use. However, the formulation of new inks for this technique is based on time-consuming trial-and-error processes to establish the optimal ink composition and printing parameters. Here, a dynamic printability window was modeled for the assessment of the printability of polysaccharide blend inks of alginate and hyaluronic acid with the intent to build a versatile predictive tool to speed up the testing procedures. The model considers both the rheological properties of the blends (viscosity, shear thinning behavior, and viscoelasticity) and their printability (in terms of extrudability and the ability of forming a well-defined filament and detailed geometries). By imposing some conditions on the model equations, it was possible to define empirical bands in which the printability is ensured. The predictive capability of the built model was successfully verified on an untested blend of alginate and hyaluronic acid chosen to simultaneously optimize the printability index and minimize the size of the deposited filament.

Severe congenital X-linked myopathy with excessive autophagy secondary to an apparently synonymous but pathogenic novel variant.

X-linked myopathy with excessive autophagy is a rare inherited disease characterized by aberrant accumulation of autophagic vacuoles in skeletal muscle. Affected males usually show a slow progression and the heart is characteristically spared. We present four male patients from the same family with an extremely aggressive form of this disease, requiring permanent mechanical ventilation from birth. Ambulation was never achieved. Three died, one in the first hour of life, one at 7 years and one at 17 years, the last death being a consequence of heart failure. Muscle biopsy showed pathognomonic features of the disease in the 4 affected males. Genetic study found a novel synonymous variant in VMA21, c.294C>T (Gly98=). Genotyping was consistent with co-segregation with the phenotype in an X-linked recessive manner. An alteration of the normal splice pattern was confirmed by transcriptome analysis, proving that the apparently synonymous variant was the cause of this extremely severe phenotype.

Results of catheter ablation with zero or near zero fluoroscopy in pediatric patients with supraventricular tachyarrhythmias.

INTRODUCTION AND OBJECTIVES: Ionizing radiation exposure in catheter ablation procedures carries health risks, especially in pediatric patients. Our aim was to compare the safety and efficacy of catheter ablation guided by a nonfluoroscopic intracardiac navigation system (NFINS) with those of an exclusively fluoroscopy-guided approach in pediatric patients. METHODS: We analyzed catheter ablation results in pediatric patients with high-risk accessory pathways or supraventricular tachycardia referred to our center during a 6-year period. We compared fluoroscopy-guided procedures (group A) with NFINS guided procedures (group B). RESULTS: We analyzed 120 catheter ablation procedures in 110 pediatric patients (11±3.2 years, 70% male); there were 62 procedures in group A and 58 in group B. We found no significant differences between the 2 groups in procedure success (95% group A vs 93.5% group B; P=.53), complications (1.7% vs 1.6%; P=.23), or recurrences (7.3% vs 6.9%; P = .61). However, fluoroscopy time (median 1.1minutes vs 12minutes; P <.0005) and ablation time (median 96.5seconds vs 133.5seconds; P=.03) were lower in group B. The presence of structural heart disease was independently associated with recurrence (P=.03). CONCLUSIONS: The use of NFINS to guide catheter ablation procedures in pediatric patients reduces radiation exposure time. Its widespread use in pediatric ablations could decrease the risk of ionizing radiation.

Amphiphilic copolymers in biomedical applications: Synthesis routes and property control.

The request of new materials, matching strict requirements to be applied in precision and patient-specific medicine, is pushing for the synthesis of more and more complex block copolymers. Amphiphilic block copolymers are emerging in the biomedical field due to their great potential in terms of stimuli responsiveness, drug loading capabilities and reversible thermal gelation. Amphiphilicity guarantees self-assembly and thermoreversibility, while grafting polymers offers the possibility of combining blocks with various properties in one single material. These features make amphiphilic block copolymers excellent candidates for fine tuning drug delivery, gene therapy and for designing injectable hydrogels for tissue engineering. This manuscript revises the main techniques developed in the last decade for the synthesis of amphiphilic block copolymers for biomedical application. Strategies for fine tuning the properties of these novel materials during synthesis are discussed. A deep knowledge of the synthesis techniques and their effect on the performance and the biocompatibility of these polymers is the first step to move them from the lab to the bench. Current results predict a bright future for these materials in paving the way towards a smarter, less invasive, while more effective, medicine.

Effect of Preoperative Infusion of Levosimendan on Biomarkers of Myocardial Injury and Haemodynamics After Paediatric Cardiac Surgery: A Randomised Controlled Trial.

OBJECTIVE: The aim was to test the hypothesis that preoperative infusion of levosimendan would decrease patients' cardiac biomarker profiles during the immediate postoperative stage (troponin I and B-type natriuretic peptide levels) more efficiently than placebo after cardiopulmonary bypass. METHODS: In a randomised, placebo-controlled, double-blinded study, 30 paediatric patients were scheduled for congenital heart disease surgery. 15 patients (50%) received prophylactic levosimendan and 15 patients (50%) received placebo from 12 h before cardiopulmonary bypass to 24 h after surgery. RESULTS: Troponin I levels were higher in the placebo group at 0, 12, and 24 h after cardiopulmonary bypass, although the mean differences between the study groups and the 95% confidence intervals (CIs) for troponin I levels did not present statistically significant differences at any of the three time points considered (mean differences [95% CIs] - 3.32 pg/ml [- 19.34 to 12.70], - 2.42 pg/ml [- 19.78 to 13.95], and - 79.94 pg/ml [- 266.99 to 16.39] at 0, 12, and 24 h, respectively). A similar lack of statistically significant difference was observed for B-type natriuretic peptide (mean differences [95% CIs] 36.86 pg/dl [- 134.16 to 225.64], - 350.79 pg/dl [- 1459.67 to 557.45], and - 310.35 pg/dl [- 1505.76 to 509.82]). Lactic acid levels were significantly lower with levosimendan; the mean differences between the study groups and the 95% CIs for lactate levels present statistically significant differences at 0 h (- 1.52 mmol/l [- 3.19 to - 0.25]) and 12 h (- 1.20 mmol/l [- 2.53 to - 0.10]) after cardiopulmonary bypass. Oxygen delivery (DO2) was significantly higher at 12 h and 24 h after surgery (mean difference [95% CI] 627.70 ml/min/m2 [122.34-1162.67] and 832.35 ml/min/m2 [58.15 to 1651.38], respectively). CONCLUSIONS: Levosimendan does not significantly improve patients' postoperative troponin I and B-type natriuretic peptide profiles during the immediate postoperative stage in comparison with placebo, although both were numerically higher with placebo. Levosimendan, however, significantly reduced lactic acid levels and improved patients' DO2 profiles. These results highlight the importance of this new drug and its possible benefit with regard to myocardial injury; however, evaluation in larger, adequately powered trials is needed to determine the efficacy of levosimendan. Trial registry number: EudraCT 2012-005310-19.

Regression of Left Ventricular Hypertrophy in a Case of Sarcomeric Hypertrophic Cardiomyopathy: An Unexpected Outcome.

We present a case of sarcomeric hypertrophy cardiomyopathy diagnosed in a child who had hypertrophy degree regression during adolescence, with no left ventricular dysfunction and no increase of the ventricular diameters. (Level of Difficulty: Intermediate.).

[Overweight and obesity in children treated for congenital heart disease]. / Sobrepeso y obesidad en niños intervenidos de cardiopatía congénita.

INTRODUCTION: The negative impact of overweight and obesity is potentially greater in children affected by a congenital heart disease (CHD). The aim of this study is to calculate the proportion of overweight and obesity in children who underwent an intervention for CHD, and to investigate systolic arterial hypertension as a possible early cardiovascular complication. PATIENTS AND METHODS: A retrospective study was conducted on patients aged 6-17 years treated for CHD, and healthy control subjects, followed-up in a Paediatric Cardiology Clinic. Body mass index percentiles were calculated according to the criteria of WHO. A review was performed on the anthropometric and clinical data, as well as the systolic blood pressure (SBP). RESULTS: A total of 440 patients were included, of which 220 had CHD. The proportion of combined obesity and overweight (body mass index percentile ≥85) was 36.4% (37.3% in healthy subjects and 35.4% in patients with CHD, P=.738). A higher prevalence of obesity (body mass index percentile ≥97) was found in CHD patients (22.7%) compared to 15.5% in healthy subjects (P=.015). SBP percentiles were higher in overweight compared to normal-weight patients (P < .001). The prevalence of SBP readings ≥ the 95th percentile was greater in overweight than in normal weight CHD patients (29.5% versus 7.7%, P < .001) and also in the overweight healthy controls compared to those of normal weight (12.2% versus 0.7%, P < .001). CONCLUSIONS: The proportion of obesity is high in children treated for CHD and it is associated with elevated SBP levels. The risk of long-term complications needs to be reduced by means of prevention and treatment of obesity in this vulnerable population.

Digenic Heterozigosity in SCN5A and CACNA1C Explains the Variable Expressivity of the Long QT Phenotype in a Spanish Family.

INTRODUCTION AND OBJECTIVES: A known long QT syndrome-related mutation in Nav1.5 cardiac channels (p.R1644H) was found in 4 members of a Spanish family but only 1 of them showed prolongation of the QT interval. In the other 3 relatives, a novel missense mutation in Cav1.2 cardiac channels was found (p.S1961N). Here, we functionally analyzed p.S1961N Cav1.2 channels to elucidate whether this mutation regulates the expressivity of the long QT syndrome phenotype in this family. METHODS: L-type calcium current (ICaL) recordings were performed by using the whole-cell patch-clamp technique in Chinese hamster ovary cells transiently transfected with native and/or p.S1961N Cav1.2 channels. RESULTS: Expression of p.S1961N channels significantly decreased ICaL density. Using Ba as a charge carrier to suppress the Ca-dependent inactivation of Cav1.2 channels, we demonstrated that the mutation significantly accelerates the voltage-dependent inactivation of Cav1.2 channels decreasing the inactivation time constant. As a consequence, the total charge flowing through p.S1961N Cav1.2 channels significantly decreased. The effects of the p.S1961N Cav1.2 and p.R1644H Nav1.5 mutations alone or their combination on the action potential (AP) morphology were simulated using a validated model of the human ventricular AP. The p.S1961N Cav1.2 mutation shortens the AP duration and abrogates the prolongation induced by p.R1644H Nav1.5 channels. CONCLUSIONS: The p.S1961N mutation in Cav1.2 channels decreased the ICaL, an effect which might shorten ventricular AP. The presence of the loss-of-function Cav1.2 mutation could functionally compensate the prolonging effects produced by the Nav1.5 gain-of-function mutation.

Genetic Mosaicism in Calmodulinopathy.

BACKGROUND: CaM (calmodulin) mutations are associated with congenital arrhythmia susceptibility (calmodulinopathy) and are most often de novo. In this report, we sought to broaden the genotype-phenotype spectrum of calmodulinopathies with 2 novel calmodulin mutations and to investigate mosaicism in 2 affected families. METHODS: CaM mutations were identified in 4 independent cases by DNA sequencing. Biochemical and electrophysiological studies were performed to determine functional consequences of each mutation. RESULTS: Genetic studies identified 2 novel CaM variants (CALM3-E141K in 2 cases; CALM1-E141V) and one previously reported CaM pathogenic variant (CALM3-D130G) among 4 probands with shared clinical features of prolonged QTc interval (range 505-725 ms) and documented ventricular arrhythmia. A fatal outcome occurred for 2 of the cases. The parents of all probands were asymptomatic with normal QTc duration. However, 2 of the families had multiple affected offspring or multiple occurrences of intrauterine fetal demise. The mother from the family with recurrent intrauterine fetal demise exhibited the CALM3-E141K mutant allele in 25% of next-generation sequencing reads indicating somatic mosaicism, whereas CALM3-D130G was present in 6% of captured molecules of the paternal DNA sample, also indicating mosaicism. Two novel mutations (E141K and E141V) impaired Ca2+ binding affinity to the C-domain of CaM. Human-induced pluripotent stem cell-derived cardiomyocytes overexpressing mutant or wild-type CaM showed that both mutants impaired Ca2+-dependent inactivation of L-type Ca2+ channels and prolonged action potential duration. CONCLUSIONS: We report 2 families with somatic mosaicism associated with arrhythmogenic calmodulinopathy, and demonstrate dysregulation of L-type Ca2+ channels by 2 novel CaM mutations affecting the same residue. Parental mosaicism should be suspected in families with unexplained fetal arrhythmia or fetal demise combined with a documented CaM mutation.

Clinical and Genetic Diagnosis of Nonischemic Sudden Cardiac Death.

INTRODUCTION AND OBJECTIVES: Nonischemic sudden cardiac death (SCD) is predominantly caused by cardiomyopathies and channelopathies. There are many diagnostic tests, including some complex techniques. Our aim was to analyze the diagnostic yield of a systematic diagnostic protocol in a specialized unit. METHODS: The study included 56 families with at least 1 index case of SCD (resuscitated or not). Survivors were studied with electrocardiogram, advanced cardiac imaging, exercise testing, familial study, genetic testing and, in some cases, pharmacological testing. Families with deceased probands were studied using the postmortem findings, familial evaluation, and molecular autopsy with next-generation sequencing (NGS). RESULTS: A positive diagnosis was obtained in 80.4% of the cases, with no differences between survivors and nonsurvivors (P=.53). Cardiac channelopathies were more prevalent among survivors than nonsurvivors (66.6% vs 40%, P=.03). Among the 30 deceased probands, the definitive diagnosis was given by autopsy in 7. A diagnosis of cardiomyopathy tended to be associated with a higher event rate in the family. Genetic testing with NGS was performed in 42 index cases, with a positive result in 28 (66.6%), with no differences between survivors and nonsurvivors (P=.21). CONCLUSIONS: There is a strong likelihood of reaching a diagnosis in SCD after a rigorous protocol, with a more prevalent diagnosis of channelopathy among survivors and a worse familial prognosis in cardiomyopathies. Genetic testing with NGS is useful and its value is increasing with respect to the Sanger method.

Calmodulin 2 Mutation N98S Is Associated with Unexplained Cardiac Arrest in Infants Due to Low Clinical Penetrance Electrical Disorders.

BACKGROUND: Calmodulin 1, 2 and 3 (CALM) mutations have been found to cause cardiac arrest in children at a very early age. The underlying aetiology described is long QT syndrome (LQTS), catecholaminergic polymorphic ventricular tachycardia (CPVT) and idiopathic ventricular fibrillation (IVF). Little phenotypical data about CALM2 mutations is available. OBJECTIVES: The aim of this paper is to describe the clinical manifestations of the Asn98Ser mutation in CALM2 in two unrelated children in southern Spain with apparently unexplained cardiac arrest/death. METHODS: Two unrelated children aged 4 and 7, who were born to healthy parents, were studied. Both presented with sudden cardiac arrest. The first was resuscitated after a VF episode, and the second died suddenly. In both cases the baseline QTc interval was within normal limits. Peripheral blood DNA was available to perform targeted gene sequencing. RESULTS: The surviving 4-year-old girl had a positive epinephrine test for LQTS, and polymorphic ventricular ectopic beats were seen on a previous 24-hour Holter recording from the deceased 7-year-old boy, suggestive of a possible underlying CPVT phenotype. A p.Asn98Ser mutation in CALM2 was detected in both cases. This affected a highly conserved across species residue, and the location in the protein was adjacent to critical calcium binding loops in the calmodulin carboxyl-terminal domain, predicting a high pathogenic effect. CONCLUSIONS: Human calmodulin 2 mutation p.Asn98Ser is associated with sudden cardiac death in childhood with a variable clinical penetrance. Our results provide new phenotypical information about clinical behaviour of this mutation.

Successful tocilizumab treatment in a child with refractory Takayasu arteritis.

Takayasu arteritis (TA) in the child remains a therapeutic challenge because corticosteroids and conventional immunosuppressive agents are not always safe or efficacious. The complex formed by interleukin-6 (IL-6) and soluble IL-6 receptor appears to play a pivotal role in the pathogenesis of TA. We describe a favorable response to the anti-IL-6 receptor antibody tocilizumab (TCZ) in a child with aggressive and refractory TA including an assessment of the proinflammatory cytokine profile. A 3-year-old girl with TA consisting of thickening of the aortic arch wall, severe obstruction of the supra-aortic branches, and complete occlusion of both common carotid arteries failed to respond to corticosteroids, methotrexate, tumor necrosis factor α blockade, cyclophosphamide, and mycophenolate mofetil, and 3 years later, the disease remained active with severe manifestations (brain ischemia). The patient underwent percutaneous angioplasty, although significant restenosis was soon documented. After a severe relapse, the patient started TCZ infusions (8 mg/kg for 2 weeks), and a rapid clinical remission was observed, associated with a drastic reduction of inflammatory markers and IL-6 levels. Corticosteroids were withdrawn, the patient's weight and height improved, and bone mineral density values returned to normal. Two years later, TCZ infusions were extended, with no significant side effects. Cerebral ischemia resolved, and recanalization of the previously occluded supra-aortic branches was performed.