Overexpression of VEGFα as a biomarker of endothelial dysfunction in aortic tissue of α-GAL-Tg/KO mice and its upregulation in the serum of patients with Fabry's disease.

Introduction: Fabry's disease is an X-linked lysosomal storage disorder caused by reduced activity of α-galactosidase A (GAL), leading to premature death on account of renal, cardiac, and vascular organ failure. Accumulation of the GAL substrate globotriaosylceramide (Gb3) in endothelial and smooth muscle cells is associated with early vascular cell damage, suggesting endothelial dysfunction as a driver of cardiorenal organ failure. Here, we studied the vascular expression of the key angiogenic factors, VEGFα and its antagonist angiostatin, in Fabry α-GAL-Tg/KO mice and determined circulating VEGFα and angiostatin serum levels in patients with Fabry's disease and healthy controls. Methods: Cryopreserved aortic vessels from six α-GAL-Tg/KO and six wild-type (WT) mice were obtained and VEGFα and angiostatin levels were determined by performing Western blot analysis. VEGFα expression was visualized by an immunohistochemical staining of paraffin aortic rings. In addition, VEGFα and angiostatin serum levels were measured by using an enzyme-linked immunosorbent assay in 48 patients with genetically verified Fabry's disease (50% male) and 22 healthy controls and correlated with disease severity markers such as lyso-Gb3, albuminuria, NTproBNP, high-sensitive troponin T (hsTNT), and myocardial wall thickness. Results: It was found that there was a significant increase in VEGFα protein expression (1.66 ± 0.35 vs. 0.62 ± 0.16, p = 0.0009) and a decrease in angiostatin expression (0.024 ± 0.007 vs. 0.053 ± 0.02, p = 0.038) in aortic lysates from α-GAL-Tg/KO compared with that from WT mice. Immunohistochemical staining revealed an adventitial VEGFα signal in α-GAL-Tg/KO mice, whereas no VEGFα signal could be detected in WT mice aortas. No differences in aortic angiostatin expression between α-GAL-Tg/KO- and WT mice could be visualized. The serum levels of VEGFα were significantly upregulated in patients with Fabry's disease compared with that in healthy controls (708.5 ± 426.3 vs. 458.5 ± 181.5 pg/ml, p = 0.048) and positively associated with albuminuria (r = 0.82, p < 0.0001) and elevated NTproBNP (r = 0.87, p < 0.0001) and hsTNT values (r = 0.41, p = 0.048) in male patients with Fabry's disease. For angiostatin, no significant difference was found between patients with Fabry's disease and healthy controls (747.6 ± 390.3 vs. 858.8 ± 599.3 pg/ml). Discussion: In conclusion, an overexpression of VEGFα and downregulation of its counter player angiostatin in aortic tissue of α-GAL-Tg/KO mice support the hypothesis of an underlying vasculopathy in Fabry's disease. Elevated VEGFα serum levels were also observed in patients with Fabry's disease and were positively associated with elevated markers of organ manifestation in males. These findings suggest that angiogenetic markers, such as VEGFα, may be potentially useful biomarkers for the detection of endothelial dysfunction in classical Fabry's disease.

Characterisation of patients referred to a tertiary-level inherited cardiac condition clinic with suspected arrhythmogenic right ventricular cardiomyopathy (ARVC).

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy is a rare inherited disease with incomplete penetrance and an environmental component. Although a rare disease, ARVC is a common cause of sudden cardiac death in young adults. Data on the different stages of ARVC remains scarce. The purpose of this study is to describe the initial presentation and cardiac phenotype of definite and non-definite ARVC for patients seen at a tertiary service. METHODS: This is a single centre, observational cohort study of patients with definite and non-definite ARVC seen at the Inherited Cardiac Conditions services at University Hospital Birmingham (UHB) in the period 2010-2021. Patients were identified by interrogation of digital health records, medical history, imaging and by examining 12-lead electrocardiograms (ECG). RESULT: The records of 1451 patients were reviewed; of those, 165 patients were at risk of ARVC (mean age 41 ± 17 years, 56% male). 60 patients fulfilled task force criteria for definite ARVC diagnosis (n = 40, 67% males), and 38 (72%) of them carried a known pathogenic variant. The remaining 105 patients (50% males) were non-definite, and of these 45 (62%) carried a known pathogenic variant. Patients in the definite group were more symptomatic, with palpitations (57% vs. 17%), syncope (35% vs. 6%) and shortness of breath (28% vs. 5%, p < 0.001). T-wave inversion in V1-V3 and epsilon waves were observed only in the definite group. Both PR interval and QRS duration were longer in the definite (170 ± 34 ms and 100 ± 19 ms, p < 0.001) compared to (149 ± 25 and 91 ± 14 ms, p = 0.005). Patients with definite ARVC had significantly larger RV end diastolic areas and significantly reduced biventricular function (RVEDA = 27 ± 10 cm2, RVFAC = 37 ± 11% and EF = 56 ± 12%) compared to the non-definite group (RVEDA = 18 ± 4 cm2, RVFAC 49 ± 6% and LVEF 64 ± 7%, p < 0.001). Sustained ventricular tachycardia (VT) occurred more frequently in the definite group compared to the non-definite group (27% vs. 2%, p < 0.001). Ventricular fibrillation was observed in the definite group only (8 of 60 patients, 13%). CONCLUSION: Our study showed differences between definite and non-definite ARVC patients in terms of clinical, electrophysiological and imaging features. Major adverse cardiac events occurred more commonly in the definite group, but also were observed in non-definite ARVC. This single centre observational cohort study forms a basis for further prospective multicentre interventional studies.

Familial atrial fibrillation mutation M1875T-SCN5A increases early sodium current and dampens the effect of flecainide.

AIMS: Atrial fibrillation (AF) is the most common cardiac arrhythmia. Pathogenic variants in genes encoding ion channels are associated with familial AF. The point mutation M1875T in the SCN5A gene, which encodes the α-subunit of the cardiac sodium channel Nav1.5, has been associated with increased atrial excitability and familial AF in patients. METHODS AND RESULTS: We designed a new murine model carrying the Scn5a-M1875T mutation enabling us to study the effects of the Nav1.5 mutation in detail in vivo and in vitro using patch clamp and microelectrode recording of atrial cardiomyocytes, optical mapping, electrocardiogram, echocardiography, gravimetry, histology, and biochemistry. Atrial cardiomyocytes from newly generated adult Scn5a-M1875T+/- mice showed a selective increase in the early (peak) cardiac sodium current, larger action potential amplitude, and a faster peak upstroke velocity. Conduction slowing caused by the sodium channel blocker flecainide was less pronounced in Scn5a-M1875T+/- compared to wildtype atria. Overt hypertrophy or heart failure in Scn5a-M1875T+/- mice could be excluded. CONCLUSION: The Scn5a-M1875T point mutation causes gain-of-function of the cardiac sodium channel. Our results suggest increased atrial peak sodium current as a potential trigger for increased atrial excitability.

Smartphone and wearable detected atrial arrhythmias in Older Adults: Results of a fully digital European Case finding study.

Aims: Simplified detection of atrial arrhythmias via consumer-electronics would enable earlier therapy in at-risk populations. Whether this is feasible and effective in older populations is not known. Methods and results: The fully remote, investigator-initiated Smartphone and wearable detected atrial arrhythmia in Older Adults Case finding study (Smart in OAC-AFNET 9) digitally enrolled participants ≥65 years without known atrial fibrillation, not receiving oral anticoagulation in Germany, Poland, and Spain for 8 weeks. Participants were invited by media communications and direct contacts. Study procedures adhered to European data protection. Consenting participants received a wristband with a photoplethysmography sensor to be coupled to their smartphone. The primary outcome was the detection of atrial arrhythmias lasting 6 min or longer in the first 4 weeks of monitoring. Eight hundred and eighty-two older persons (age 71 ± 5 years, range 65-90, 500 (57%) women, 414 (47%) hypertension, and 97 (11%) diabetes) recorded signals. Most participants (72%) responded to adverts or word of mouth, leaflets (11%) or general practitioners (9%). Participation was completely remote in 469/882 persons (53%). During the first 4 weeks, participants transmitted PPG signals for 533/696 h (77% of the maximum possible time). Atrial arrhythmias were detected in 44 participants (5%) within 28 days, and in 53 (6%) within 8 weeks. Detection was highest in the first monitoring week [incidence rates: 1st week: 3.4% (95% confidence interval 2.4-4.9); 2nd-4th week: 0.55% (0.33-0.93)]. Conclusion: Remote, digitally supported consumer-electronics-based screening is feasible in older European adults and identifies atrial arrhythmias in 5% of participants within 4 weeks of monitoring (NCT04579159).

JavaCyte, a novel open-source tool for automated quantification of key hallmarks of cardiac structural remodeling.

Many cardiac pathologies involve changes in tissue structure. Conventional analysis of structural features is extremely time-consuming and subject to observer bias. The possibility to determine spatial interrelations between these features is often not fully exploited. We developed a staining protocol and an ImageJ-based tool (JavaCyte) for automated histological analysis of cardiac structure, including quantification of cardiomyocyte size, overall and endomysial fibrosis, spatial patterns of endomysial fibrosis, fibroblast density, capillary density and capillary size. This automated analysis was compared to manual quantification in several well-characterized goat models of atrial fibrillation (AF). In addition, we tested inter-observer variability in atrial biopsies from the CATCH-ME consortium atrial tissue bank, with patients stratified by their cardiovascular risk profile for structural remodeling. We were able to reproduce previous manually derived histological findings in goat models for AF and AV block (AVB) using JavaCyte. Furthermore, strong correlation was found between manual and automated observations for myocyte count (r = 0.94, p < 0.001), myocyte diameter (r = 0.97, p < 0.001), endomysial fibrosis (r = 0.98, p < 0.001) and capillary count (r = 0.95, p < 0.001) in human biopsies. No significant variation between observers was observed (ICC = 0.89, p < 0.001). We developed and validated an open-source tool for high-throughput, automated histological analysis of cardiac tissue properties. JavaCyte was as accurate as manual measurements, with less inter-observer variability and faster throughput.

Minimally invasive closed-chest ultrasound-guided substance delivery into the pericardial space in mice.

Organ-directed gene transfer remains an attractive method for both gaining a better understanding of heart disease and for cardiac therapy. However, virally mediated transfer of gene products into cardiac cells requires prolonged exposure of the myocardium to the viral substrate. Pericardial injection of viral vectors has been proposed and used with some success to achieve myocardial transfection and may be a suitable approach for transfection of atrial myocardium. Indeed, such an organ-specific method would be particularly useful to reverse phenotypes in young and adult genetically altered murine models of cardiac disease. We therefore sought to develop a minimally invasive technique for pericardial injection of substances in mice. Pericardial access in anaesthetised, spontaneously breathing mice was achieved using continuous high-resolution ultrasound guidance. We could demonstrate adequate delivery of injected substances into the murine pericardium. Atrial epicardial and myocardial cells were transfected in approximately one third of mice injected with enhanced green fluorescent protein-expressing adenovirus. Cellular expression rates within individual murine atria were limited to a maximum of 20 %; therefore, expression efficiency needs to be further improved. Minimally invasive, ultrasound-guided injection of viral material appears a technically challenging yet feasible method for selective transfection of atrial epi- and myocardium. This pericardial injection method may be useful in the evaluation of potential genetic interventions aimed at rescuing atrial phenotypes in transgenic mouse models.

Gene construction, expression and functional testing of an inotropic peptide from the venom of the black scorpion Hottentotta judaicus.

Anti-insect depressant toxins represent a subfamily of scorpion venom-derived ß-toxins that are polypeptides composed of 61-65 amino acid residues stabilized by four disulfide bridges. These toxins affect the activation of voltage-sensitive sodium channels (NaScTx) and exhibit the preferential ability to induce flaccid paralysis in insect larvae. Here we demonstrate the recombinant expression of the novel cardiac inotropic peptide (Bj-IP) that was classified as an anti-insect depressant ßNaScTx isolated from the venom of Hottentotta judaicus. By using "splicing by overlap extension" (SOE)-PCR, allowing for the first time one step de novo synthesis of long-chain scorpion toxin genes, we generated a codon-optimized DNA fragment of Bj-IP for cloning into the Escherichia coli vector pQE30. Moreover, the gene of interest was fused to a 6xHis coding DNA sequence. Subsequent recombinant expression was performed in E. coli KRX. The purification of the polypeptide was achieved by a combination of NiNTA agarose columns and RP (C(18)) high-performance liquid chromatography. The purified fusion protein was digested with factor Xa resulting in the elution of Bj-IP. The yield of recombinant Bj-IP expression was approximately 4.5 mg per liter of culture. Mass spectrometry confirmed the theoretical total mass of Bj-IP (6608 Da). Tag-free Bj-IP was refolded in guanidine chloride buffer with a glutathione redox system which was supplemented with different additives at 16 °C. Supplementation with 10% glycerol produced Bj-IP folding forms that exhibited reproducible biological activity in mouse cardiomyocytes. Cell contractility was increased by almost 3-fold and decay kinetics were hasten by 47% after administration of Bj-IP. Taken together, here we show the recombinant expression of the functionally active cardiac inotropic peptide Bj-IP, a new ßNaScTx from H. judaicus, for promising pharmacological applications. Furthermore, our data suggest that the use of SOE-PCR may help to facilitate in future the high throughput of cloning and/or modification of scorpion toxin genes.

Arrhythmogenic right ventricular cardiomyopathy: an update on pathophysiology, genetics, diagnosis, and risk stratification.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiomyopathy accounting for life-threatening ventricular tachyarrhythmias and sudden death in young individuals and athletes. Over the past years, mutations in desmosomal genes have been identified as disease-causative. However, genetic heterogeneity and variable phenotypic expression alongside with diverse disease progression still render the evaluation of its prognostic implication difficult. ARVC was initially entered into the canon of cardiomyopathies of the World Health Organization in 1995, and international efforts have resulted in the 2010 modified diagnostic criteria for ARVC. Despite all additional insights into pathophysiology, clinical management, and modern risk stratification, under-/misdiagnosing of ARVC remains a problem and hampers reliable statements on the incidence, prevalence, and natural course of the disease.This review provides a comprehensive overview of the current literature on the pathogenesis, diagnosis, treatment, and prognosis of ARVC and sheds some light on potential new developments in these areas.

Preclinical testing of drug-induced proarrhythmia: value of transgenic models.

Drug-induced proarrhythmia is a serious medical problem that causes relevant morbidity and mortality. It is also a relevant problem for the development of novel pharmacological compounds. Therefore, there is a need for sensitive, specific and high-throughput preclinical tests to detect a risk for drug-induced proarrhythmia early in the development of new drugs. The review focuses on the potential role of transgenic models with altered repolarisation but without overt structural heart disease for drug-induced proarrhythmia screening. Today, selected murine models with alterations in K+, Na+ channels and ankyrin are available. In the future, transgenic rabbit and Zebra fish models may also be used.

Leukocyte adhesion deficiency II syndrome, a generalized defect in fucose metabolism.

Leukocyte adhesion deficiency II has been described in only 2 patients; herein we report extensive investigation of another patient. The physical stigmata were detected during prenatal ultrasonographic investigation. Sialyl-Lewis X (sLex) was absent from the surface of polymorphonuclear neutrophils, and cell binding to E- and P-selectin was severely impaired, causing an immunodeficiency. The elevation of peripheral neutrophil counts occurred within several days after birth. A severe hypofucosylation of glycoconjugates bearing fucose in different glycosidic links was present in all cell types investigated, demonstrating that leukocyte adhesion deficiency II is not only a disorder of leukocytes but a generalized inherited metabolic disease affecting the metabolism of fucose.