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Aims: Rare, deleterious genetic variants in FLT4 are associated with Tetralogy of Fallot (TOF), the most common cyanotic congenital heart disease. The distinct genetic variants in FLT4 are also an established cause of Milroy disease, the most prevalent form of primary hereditary lymphoedema. The phenotypic features of these two conditions are non-overlapping, implying pleiotropic cellular mechanisms during development., Methods and Results: In this study, we show that FLT4 variants identified in patients with TOF, when expressed in primary human endothelial cells, cause aggregation of FLT4 protein in the perinuclear endoplasmic reticulum, activating proteostatic and metabolic signalling, whereas lymphoedema-associated FLT4 variants and wild-type (WT) FLT4 do not. FLT4 TOF variants display characteristic gene expression profiles in key developmental signalling pathways, revealing a role for FLT4 in cardiogenesis distinct from its role in lymphatic development. Inhibition of proteostatic signalling abrogates these effects, identifying potential avenues for therapeutic intervention. Depletion of flt4 in zebrafish caused cardiac phenotypes of reduced heart size and altered heart looping. These phenotypes were rescued with coinjection of WT human FLT4 mRNA, but incompletely or not at all by mRNA harbouring FLT4 TOF variants., Conclusion: Taken together, we identify a pathogenic mechanism for FLT4 variants predisposing to TOF that is distinct from the known dominant negative mechanism of Milroy-causative variants. FLT4 variants give rise to conditions of the two circulatory subdivisions of the vascular system via distinct developmental pleiotropic molecular mechanisms., Competing Interests: Conflict of interest: none declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Objective: Left ventricular assist devices (LVAD) have been used to 'bridge' patients with end-stage heart failure to transplantation. Although several reports have suggested that the native ventricular function recovers after long-term LVAD support, a process called 'reverse remodeling', the underlying biological mechanisms are still unknown. As the transcription factor nuclear factor-kappaB (NF-kappaB) has been shown to be active in the failing human heart, we examined whether its activity is altered under LVAD support, and may thus contribute to the dynamic process of 'reverse remodeling'., Methods: The activity of NF-kappaB was studied in 16 patients with end-stage heart failure (eight with dilated cardiomyopathy, six with ischemic heart disease, one with myocarditis, and one with congenital heart disease) before and after LVAD support by immunohistochemistry using an antibody against active NF-kappaB. Gel-shifts for NF-kappaB DNA-binding activity were performed with paired human myocardial tissue from four patients. The mean cardiomyocyte diameter before and after mechanical unloading was measured with an image analyzer system., Results: 15 patients out of 16 showed a significant decrease in the number of NF-kappaB positive cardiomyocyte nuclei after LVAD support in the left ventricular myocardium. The NF-kappaB DNA-binding activity also decreased after LVAD support as measured by gel-shift analysis. While the number of positive cardiomyocytes was significantly higher in the subendocardium than in the subepicardium at the time of LVAD implantation, this difference was no longer present at the time of LVAD explantation. The diameter of cardiomyocytes in the left ventricle decreased significantly as a parameter of structural reverse remodeling., Conclusion: LVAD support decreases the extent of NF-kappaB activation in failing human hearts, suggesting that NF-kappaB may be involved in the process of 'reverse remodeling'.

Objective: The aim was to investigate the electrophysiological properties of the L-type calcium current (ICa,L) in ventricular myocytes at a physiological temperature (36-37 degrees C) isolated from children undergoing surgical repair of tetralogy of Fallot., Methods: ICa,L was recorded with the patch-clamp technique in the single electrode whose-cell mode at a physiological calcium concentration (1.8 mmol/l) at 36-37 degrees C., Results: Under these conditions, maximum current density averaged -5.80 +/- 0.45 pA/pF. ICa,L showed a bell-shaped current-voltage relationship: the current activated at -37.7 +/- 1.36 mV, peaked at +9.41 +/- 1.60 mV and reversed at +57.7 +/- 2.12 mV (n = 17). At +10 mV, time to peak of ICa,L was 5.23 +/- 0.46 ms. Membrane potentials for half-maximal steady-state activation and inactivation of ICa,L were -6.02 and -20.4 mV, respectively, the slope factors were 7.16 mV for steady-state activation and 6.49 mV for steady-state inactivation. ICa,L did not completely inactivate and showed a big window current between -45 and +40 mV. The inactivation of ICa,L showed a biexponential time course with a fast time constant ranging from 9.11 to 12.9 ms and a slow time constant ranging from 60.9 to 220 ms between -30 and +30 mV. Only the slow time constant showed a pronounced voltage dependency. The recovery from inactivation of ICa,L was biphasic with a fast time constant of 60.7 ms and a slow time constant of 619 ms. beta-Adrenergic stimulation with isoprenaline (1 mumol/l) increased the ICa,L density from -5.71 +/- 1.55 to -13.8 +/- 1.96 pA/pF (142%; P < 0.05) at +10 mV., Conclusions: The present study demonstrates that most of the electrophysiological properties of ICa,L in ventricular myocytes isolated from children with tetralogy of Fallot resemble those of adult ventricular cells. The existence of a big calcium window current could be involved in the occurrence of early afterdepolarizations which could lead to the high incidence of arrhythmias after surgical repair of tetralogy of Fallot.

Myosin isotype composition was examined in ventricular infundibular muscle from 40 patients with tetralogy of Fallot, aged from 7 months to 38 years. Results were compared with normal samples of ventricular infundibulum from subjects in the same age range, from 18-20 week old fetuses to 6 month old neonates, and from 43-81 year old adults. Myosin light chain isotypes were examined by one dimensional and two dimensional gel electrophoresis and quantified densitometrically. Heavy chain isotypes were examined by electrophoresis of whole heavy chains and peptide mapping after limited proteolytic digestion with chymotrypsin. At mid-gestation in normal tissues, only ventricular light chain 2 was present but light chain 1 consisted almost equally of atrial and ventricular isotypes. Amounts of atrial light chain 1 declined towards birth and disappeared during the first year after birth, gradually being replaced by ventricular light chain 1. Relative amounts of total light chains 1 and 2 remained equal. In tetralogy of Fallot atrial light chain 1 expression did not cease neonatally with mean values of 11.8% of total light chain 1 present between 7 months and 2 years, decreasing to 1.7% at 6.5-12 years and then increasing again to 3.4% in adults. A value of 34% atrial light chain 1 was present in one subject. As in normal subjects, equimolar amounts of total light chains 1 and 2 were retained. No evidence of new light chain isotypes was found in tetralogy of Fallot. Heavy chain expression was constant in normal infundibulum with only beta-heavy chain (V3 isozyme) present in the fetus, neonate, and adult.(ABSTRACT TRUNCATED AT 250 WORDS)