Acute cellular rejection (ACR) in heart transplant (HTx) recipients may be accompanied by cardiac cell damage with subsequent exposure to cardiac autoantigens and the production of cardiac ...autoantibodies (aABs). This study aimed to evaluate a peptide array screening approach for cardiac aABs in HTx recipients during ACR (ACR-HTx).
In this retrospective single-center observational study, sera from 37 HTx recipients, as well as age and sex-matched healthy subjects were screened for a total of 130 cardiac aABs of partially overlapping peptide sequences directed against structural proteins using a peptide array approach.
In ACR-HTx, troponin I (TnI) serum levels were found to be elevated. Here, we could identify aABs against beta-2-adrenergic receptor (β-2AR: EAINCYANETCCDFFTNQAY) to be upregulated in ACR-HTx (intensities: 0.80 versus 1.31, P = 0.0413). Likewise, patients positive for β-2AR aABs showed higher TnI serum levels during ACR compared with aAB negative patients (10.0 versus 30.0 ng/L, P = 0.0375). Surprisingly, aABs against a sequence of troponin I (TnI: QKIFDLRGKFKRPTLRRV) were found to be downregulated in ACR-HTx (intensities: 3.49 versus 1.13, P = 0.0025). A comparison in healthy subjects showed the same TnI sequence to be upregulated in non-ACR-HTx (intensities: 2.19 versus 3.49, P = 0.0205), whereas the majority of aABs were suppressed in non-ACR-HTx.
Our study served as a feasibility analysis for a peptide array screening approach in HTx recipients during ACR and identified 2 different regulated aABs in ACR-HTx. Hence, further multicenter studies are needed to evaluate the prognostic implications of aAB testing and diagnostic or therapeutic consequences.
Abstract only Objective: Neuregulin-1 (Nrg1) promotes cardiomyocyte hypertrophy, survival, and cell cycle activity through ErbB signaling. It is investigated in clinical trials as a cardioprotective ...agent; however, its therapeutic use might be limited due to its pro-neoplastic potential for non-cardiomyocytes that makes targeted approaches necessary. High-throughput screening of hypertrophic agonists found that Nrg1 induces CITED4 (C4) expression. C4 is also upregulated in both physiological and pathological cardiac growth and necessary to prevent adverse remodeling in vivo. However, how C4 regulates Nrg1-signaling in the heart is yet unknown. Methods: We combined pulsed-SILAC labeling, click-chemistry and mass spectrometry that allows to capture immediate changes in the proteome and secretome after Nrg1 (12h and 24h) stimulation in siRNA-mediated C4-knockdown (C4KD) and control (ctr) NRVM and complemented this with established cell- and molecular biology techniques to validate and investigate cellular function and molecular signaling. Results: We confirmed dose-dependent C4 mRNA upregulation in response to Nrg1 stimulation in NRVM. Nrg1 downstream signaling through AKT was hindered in C4KD. Computational analysis comparing the nascent proteome after Nrg1 stimulation in C4KD and ctr NRVM revealed that C4 significantly regulates proteins responsible for translation, RNA processing and energy derivation. Consistent with previously observed development of cardiac fibrosis in C4 knockout mice in vivo , we found significant upregulation of TGFb2 in C4KD NRVM. We next confirmed the upregulation of TGFb2 and its downstream effectors in C4KD and ctr, while adenoviral overexpression of C4 led to reduced TGFb2 expression. Additionally, we found TGFb2 secretion increased from C4KD NRVM with Nrg1 stimulation in the nascent secretome. Conditioned media from C4KD NRVM led to an increased pro-fibrotic response in cardiac fibroblasts. In conclusion, nascent proteomics and secretomics help to elucidate C4-dependent Nrg1-signaling, which may be an important contribution toward our goal to identify targetable cardioprotective pathways in the heart.
Animals display selective escape behaviors when faced with environmental threats. Selection of the appropriate response by the underlying neuronal network is key to maximizing chances of survival, ...yet the underlying network mechanisms are so far not fully understood. Using synapse-level reconstruction of the Drosophila larval network paired with physiological and behavioral readouts, we uncovered a circuit that gates selective escape behavior for noxious light through acute and input-specific neuropeptide action. Sensory neurons required for avoidance of noxious light and escape in response to harsh touch, each converge on discrete domains of neuromodulatory hub neurons. We show that acute release of hub neuron-derived insulin-like peptide 7 (Ilp7) and cognate relaxin family receptor (Lgr4) signaling in downstream neurons are required for noxious light avoidance, but not harsh touch responses. Our work highlights a role for compartmentalized circuit organization and neuropeptide release from regulatory hubs, acting as central circuit elements gating escape responses.
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•Connectome of a neuromodulatory circuit required for noxious light avoidance•Domain-specific input of noxious light and touch circuits on modulatory hub neurons•Acute neuropeptide release from hub neurons gates noxious light avoidance•Noxious light and touch are differentiated by selective peptide-responsive circuits
Animals escape from danger using stimulus-specific responses. Imambocus et al. show that in Drosophila larvae, neuromodulatory hub neurons help to discriminate noxious stimuli and facilitate specific behavioral responses by acute neuropeptide release to promote avoidance of noxious light.
Data on the prognostic impact of residual tricuspid regurgitation (TR) after tricuspid transcatheter edge-to-edge repair (T-TEER) are scarce. The aim of this analysis was to evaluate 2-year survival ...and symptomatic outcomes of patients in relation to residual TR after T-TEER.
Using the large European Registry of Transcatheter Repair for Tricuspid Regurgitation (EuroTR registry) we investigated the impact of residual TR on 2-year all-cause mortality and New York Heart Association (NYHA) functional class at follow-up. The study further identified predictors for residual TR ≥3+ using a logistic regression model. The study included a total of 1286 T-TEER patients (mean age 78.0 ± 8.9 years, 53.6% female). TR was successfully reduced to ≤1+ in 42.4%, 2+ in 40.0% and 3+ in 14.9% of patients at discharge, while 2.8% remained with TR ≥4+ after the procedure. Residual TR ≥3+ was an independent multivariable predictor of 2-year all-cause mortality (hazard ratio 2.06, 95% confidence interval 1.30-3.26, p = 0.002). The prevalence of residual TR ≥3+ was four times higher in patients with higher baseline TR (vena contracta >11.1 mm) and more severe tricuspid valve tenting (tenting area >1.92 cm
). Of note, no survival difference was observed in patients with residual TR ≤1+ versus 2+ (76.2% vs. 73.1%, p = 0.461). The rate of NYHA functional class ≥III at follow-up was significantly higher in patients with residual TR ≥3+ (52.4% vs. 40.5%, p < 0.001). Of note, the degree of TR reduction significantly correlated with the extent of symptomatic improvement (p = 0.012).
T-TEER effectively reduced TR severity in the majority of patients. While residual TR ≥3+ was associated with worse outcomes, no differences were observed for residual TR 1+ versus 2+. Symptomatic improvement correlated with the degree of TR reduction.
G protein-coupled receptor kinase 2 (GRK2), which is markedly upregulated in failing human myocardium, has been implicated as a contributing factor or consequence of heart failure (HF). Importantly, ...cardiac-specific GRK2 knockout mice have recently proved the pathological nature of GRK2 in HF. Targeted inhibition of GRK2 is possible using a peptide inhibitor known as the βARKct, which has rescued several disparate small animal HF models. This study was designed to evaluate long-term βARKct expression in a clinically relevant large animal HF model, using stable myocardial gene delivery with adeno-associated virus serotype 6 (AAV6).
A porcine model of HF subsequent to left ventricular (LV) myocardial infarction (MI) was used to study the effects of retrograde injection into the anterior interventricular vein of either AAV6.βARKct or AAV6.luciferase as a control 2 weeks after MI. Echocardiography and LV hemodynamics were performed before and 6 weeks after gene transfer. Robust and long-term βARKct expression was found after AAV6-mediated delivery, leading to significant amelioration of LV haemodynamics and contractile function in HF pigs compared with AAV6.luciferase-treated control animals that showed a continued decline in cardiac function. Interestingly, the neurohormonal axis was virtually normalized in AVV6.βARKct-treated HF animals, represented by reductions in plasma norepinephrine levels, whereas AAV6.luciferase-treated pigs showed further increases in plasma catecholamine levels. As a result, LV remodelling and foetal gene expression was reversed by AVV6.βARKct gene therapy.
These data--showing sustained amelioration of cardiac function in a post-MI pig HF model--demonstrate the therapeutic potential of βARKct gene therapy for HF.
Introduction: The long-term evolution of clinical, echocardiographic, and laboratory parameters of cardiac function in patients with chronic heart failure (HF) with either reduced (HFrEF) or mildly ...reduced (HFmrEF) left ventricular ejection fraction (LVEF) is incompletely characterised. Methods: We identified patients with chronic stable HF who presented at least twice to a university HF outpatient clinic between 1995 and 2021. Trajectories of NYHA functional class, LVEF, left ventricular internal end-diastolic diameter (LVIDD), NT-proBNP concentrations, and HF treatment over 10 years of follow-up were analysed using fractional polynomials. Analyses were repeated after stratifying patients according to aetiology (ischaemic vs. dilated) or HF category (HFrEF vs. HFmrEF). Results: A total of 2,132 patients were included, of whom 51% had ischaemic and 49% had dilated HF. Eighty six percent and 14% were classified as HFrEF and HFmrEF, respectively. Mean LVEF was 28 ± 10%, and median NT-proBNP and estimated glomerular filtration rate values were 1,170 (385–3,176) pmol/L and 81 (62–100) mL/min/1.73 m 2 , respectively. Median follow-up was 5.2 (2.6–9.2) years. Overall, NYHA functional class and LVIDD trajectories were U-shaped, whereas LVEF and NT-proBNP concentrations markedly improved during the first year and remained stable thereafter. However, the evolution of HF parameters significantly differed with respect to HF category and aetiology, with greater improvements seen in patients with HFrEF of non-ischaemic origin. Improvements in HF variables were associated with optimization of HF therapy, notably with initiation and up-titration of renin-angiotensin-system blockers. Conclusion: This study provides insights into the natural history of HF in a large cohort of well-treated chronic HF outpatients with respect to subgroups of HF and different aetiologists.
Abstract only Introduction: For S100A1-based heart failure gene therapies, AAV9 and 6 have shown efficacy in pre-clinical large animal studies. As AAV9 has shown concerning signs of toxicity in ...clinical studies and AAV6 displays poor production yields, there is need for a novel safe and cardiac-specific AAV serotype. Hypothesis: We hypothesized that in a pig model the safety proven and scalably manufacturable AAV5 may be a suitable vector for S100A1-based gene therapy of post-ischemic cardiac dysfunction. Methods: AAV production, 2h balloon-occlusion of the LCX, retrograde cardiac gene delivery, cardiac MRI, late gadolinium enhancement (LGE), global T1 relaxation, qPCR, RNA-Seq, WGCNA, KEGG, Reactome, LAD-ligation mouse model Results: In a comparative study of AAV5-, 6- and 9-luciferase (luc) in healthy farm pigs (n=5 each; 1x10 13 vgc/pig), AAV5 achieved a more homogeneous cardiac apical-basal transduction pattern than AAV6 with a higher luc activity than AAV9. In a clinically relevant endpoint driven study, we demonstrated a significant improvement in EF (+19 ± 5 %) 12 weeks after retrograde AAV5- S100A1 gene delivery compared to AAV5-luc in infarcted pigs (n=4 each; 1x10 13 vgc/pig). Moreover, S100A1 -treated pigs showed significantly less infarct extension (-0.5 ± 0.3 g vs. 5 ± 1.3 g (luc)) measured by cardiac MRI. There were no unfavorable alterations in blood chemistry or ECG. S100A1 expression was predominantly contained to the heart. The WGCNA unveiled a significant correlation between the improved EF and a suppression of inflammatory and immunological pathways (r=-0.96, p < 0.01) and between the absent infarct extension and enhanced activity of cardioprotective signaling (r=-0.82, p < 0.05). With injections of 2х10 11 vgc of AAV5- S100A1 or AAV5-gfp (n=4 each) into the remote myocardium in the mouse model, we confirmed a significant improvement in FS (+43.8 ± 8.8 %, vs. gfp) and suppression of inflammatory gene expression including i.e., IL1b or TNFa by S100A1. Conclusion: We conclude that AAV5 is suitable for S100A1-based gene therapy of post-ischemic cardiac dysfunction and that this vector/target combination can help accelerating the way towards a clinical trial. We also found novel signaling pathways that may be involved in S100A1’s therapeutic actions.
Abstract only Introduction: A high transduction efficacy and potency of AAV-based cardiac gene therapies is key for the clinical translation in which hereditary and acquired cardiac disorders will be ...targeted. As such, ultrapure vectors with superior biological and therapeutic capabilities are a must for these therapies. Hypothesis: We hypothesized that our developed affinity chromatography (AC) based purification system will increase AAV recovery and - potency. Methods: AAV vector production, AC based - or iodixanol density gradient (DG) based purification, Q-PCR, WB, EM, LC-MS/MS, P-loop Results: Using the same vector input quantity, the AC-based purification enabled an approximately 13-fold greater vector genome copy (vgc) recovery than the DG-based purification. Mass spectrometry analysis demonstrated ultrapure AAV9 vectors as a result of an AC purification (AAV9 93.24% vs. 6,76% contaminants) whereas corresponding DG preparations resulted in highly contaminated vectors (AAV9 5.49% vs. 94,51% contaminants). Biological potency of AC- and DG-purified AAV9 vectors towards cardiac transduction were determined by systemic injections of 1·10 10 , 1·10 11 or 1·10 12 vgc of AAV9-EGFP in C57BL/6 mice (n=8 each group). AC-purified AAVs achieved a significantly higher cardiac transduction efficacy for every dosage assessed by comparative bulk myocardial DNA, RNA and protein level analysis after 2 weeks. Therapeutic potency was examined for a recently published novel target for chronic heart failure namely the RFXP1-RLN system. To this end, a dosage of 5·10 11 vgcs of either AC- or DG purified AAV9-RXFP1 vectors were systemically injected and the cardiac contractile performance increase was captured after 2 weeks in mice of both groups. Of note, 10 minutes after RLN administration, the rise in LV +dp/dt max. was already significantly greater in the AC- than the DG-vector treated group (AC: 13594+/-1972 vs. DC: 9822+/-801 mmHg/s; n=8 per group, p<0.01). Conclusion: The data clearly promote AC-based AAV purification as a novel standard for cardiovascular basic and translational research. Higher consistency in results, higher therapeutic effects and superior biological potency can be expected from higher production yields of ultrapure AAVs even at lower vector dosages.
In Drosophila, a dedicated olfactory channel senses a male pheromone, cis-vaccenyl acetate (cVA), promoting female courtship while repelling males. Here, we show that separate cVA-processing streams ...extract qualitative and positional information. cVA sensory neurons respond to concentration differences in a 5-mm range around a male. Second-order projection neurons encode the angular position of a male by detecting inter-antennal differences in cVA concentration, which are amplified through contralateral inhibition. At the third circuit layer, we identify 47 cell types with diverse input-output connectivity. One population responds tonically to male flies, a second is tuned to olfactory looming, while a third integrates cVA and taste to coincidentally promote female mating. The separation of olfactory features resembles the mammalian what and where visual streams; together with multisensory integration, this enables behavioral responses appropriate to specific ethological contexts.
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•cVA male pheromone has a 5-mm signaling range, activating 2 parallel central pathways•Pheromone neurons have spatial receptive fields sharpened by contralateral inhibition•Position (where) and identity (what) are separated at the 3rd layer of cVA processing•Integrating taste and cVA in sexually dimorphic aSP-g controls female receptivity
An active contrast circuit effectively allows flies to “see” each other using smell.
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