Netherlands Heart Institute

Increased levels of α-tubulin and its post-translational modifications (PTMs) are found in human heart failure and could initiate diastolic dysfunction by modulating cardiomyocyte stiffness. How these modifications occur and how they may underlie cardiac dysfunction remains unknown. Upstream kinases may play a critical role, but this has not been explored. Here we address this question by, for the first time ever, determining levels of the enzymes involved in microtubule (MT) detyrosination and acetylation (αTAT1, HDAC6) in a well-characterized cohort of patients with hypertrophic cardiomyopathy (HCM). In HCM patients (N=10-11), protein levels of detyrosination enzymes remain unaltered, whilst levels of αTAT1 and HDAC6 were decreased and increased, respectively. Phosphoproteomics in HCM (N=24) and control (N=8) myocardium identified significant differences in over 1900 serine/threonine and 160 tyrosine phosphosites, in addition to increased EGFR/IGF1R-MAPK signaling in HCM. We subsequently showed that MT repolymerization was increased in HCM MYBPC3 Arg943X hiPSC-CMs. Isoprenaline-mediated PKA activation decreased MT repolymerization in hiPSC-CMs and revealed CLASP1 , MAST4 and MAP1A as potential MT modifiers in HCM. We show that the altered HCM MT code cannot be attributed to levels of key MT-modifying enzymes. By combining kinome analyses in human HCM hearts with hiPSC-CM studies on MT dynamics, PTMs and contractility we unveiled a regulatory role for MTs in the cardiomyocyte response to beta-adrenergic receptor stimulation. Disease-mediated changes in the MT code thereby exert both a direct, and indirect effect on cardiac function via mediating the response to adrenergic activation. Graphical Abstract created with BioRender.com.

Atrial fibrillation (AF) recurrence after catheter ablation (CA), often due to incomplete lesions, remains a challenge. In this study, we evaluate the role of cardiac magnetic resonance (CMR) in characterizing lesion formation and guiding re-ablation strategies in patients with paroxysmal AF undergoing radiofrequency catheter ablation (RFCA). This prospective, single-center study enrolled patients undergoing first-time RFCA for paroxysmal AF. Pre-ablation CMR was performed for functional, anatomical, and fibrosis assessment. Repeat CMR 3 months post-ablation was performed to quantify fibrosis using Image Intensity Ratio (IIR) and detect lesion gaps, defined as ≥ 3mm discontinuities in late gadolinium enhancement (LGE). AF recurrence was monitored for 18 months. 25 patients (20% female) were enrolled. 8/25 patients (32%) experienced AF recurrence. Post-ablation CMR showed significantly increased atrial fibrosis, though only in 2/25 patients (8%) complete circumferential pulmonary vein (PV) lesions were observed. Fibrosis burden did not differ significantly between patients with and without recurrence. Among 6 patients undergoing repeat ablation, 3 with LGE-defined gaps in circumferential lesions were successfully treated with a single re-isolation procedure. In contrast, 3 patients with extensive lesions and minimal or no gaps required multiple repeat procedures and additional ablation at extra-pulmonary vein sites. CMR can be a useful tool to assess post-ablation lesions and detect gaps after RFCA for paroxysmal AF. This may help distinguish recurrence due to insufficient initial ablation from other arrhythmogenic triggers, thereby guiding tailored and effective re-ablation strategies.

Solid organ transplant (SOT) recipients face elevated tuberculosis risk, yet optimal prevention strategies in low- to medium-incidence regions remain unclear. We conducted a multicenter retrospective cohort study of adult SOT recipients transplanted between 2007 and2012 at 15 European centers, with follow-up through 2018. The primary outcome was microbiologically confirmed post-transplant tuberculosis. Incidence rates were calculated per 100,000 person-years; standardized incidence ratios (SIRs) used World Health Organization country-specific background rates. Cox models assessed risk factors. Among 5805 patients (median age 51; 62.7% male; 73.9% renal transplants), 33.8% were tested for tuberculosis infection and 10.3% received tuberculosis preventive therapy (TPT). Over 33,785 person-years, 23 patients (0.4%) developed tuberculosis (68.0/100,000 person-years). Highest incidence occurred in patients with positive screening but no TPT (233.8/100,000). Incidence was higher in Southern vs. Central Europe (251.9 vs. 28.7/100,000), with pooled SIRs of 12.8 and 3.1, respectively. Tuberculosis risk was elevated among Southern European recipients (HR 22.9) and those with migration history (HR 2.7). Tuberculosis risk is increased in European SOT recipients. Regionally adapted prevention strategies, including targeted screening in low-incidence areas and universal screening in higher-incidence regions, are warranted.

BackgroundAs survival rates after allogeneic stem cell transplantation (alloSCT) continue to grow, cardiovascular disease is becoming a major complication after alloSCT. However, the prognostic value of cardiac biomarkers has not been widely investigated.ObjectivesThis study aims to investigate the association between baseline N-terminal prohormone of brain natriuretic peptide (NT-proBNP) in patients treated with alloSCT and mortality.MethodsPatients who were referred for alloSCT between 2012 and 2023 were included in this single-center retrospective cohort study if NT-proBNP was measured <180 days before alloSCT. The primary study outcome was all-cause mortality and the secondary study outcome was a composite endpoint of major adverse cardiac events (acute coronary syndrome, device implantation, arrhythmias, heart failure, and pericarditis).ResultsWe included 807 patients, of whom 34.5% were female with a median age of 58.6 (47.9-66.0) years who had a median NT-proBNP at baseline of 113.2 (56.9-229.4) ng/L. During follow-up of 1.6 (0.6-4.0) years, 393 patients (48.7%) died, of whom 61.8% due to a complication of alloSCT. Patients who died had a significant higher baseline NT-proBNP compared to survivors (122.4 [66.4-273.6] ng/L vs 100.2 [51.2-214.2] ng/L, P = 0.001). Patients with NT-proBNP ≥125 ng/L showed a lower 5-year survival rate (P = 0.0035). In a multivariable Cox regression model, log-transformed NT-proBNP (HR: 1.21 [95% CI: 1.11-1.31], P < 0.001) and NT-proBNP ≥125 ng/L (HR: 1.29 [95% CI: 1.04-1.60], P = 0.020) were positively associated with increased risk of death.ConclusionsBaseline NT-proBNP in patients treated with alloSCT is positively associated with an increased risk of all-cause mortality. NT-proBNP ≥125 ng/L was associated with a significant lower 5-year survival.

Abstract Background/ Objectives The PLN-R14del mutation is a founder mutation originating in the Netherlands approximately 600-800 years ago and is identified in 10-15% of patients with dilated cardiomyopathy (DCM) or arrhythmogenic cardiomyopathy (ACM)(1). PLN regulates cardiomyocyte calcium handling by acting as a primary inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+ATPase 2a (SERCA2a). The PLN-R14del mutation disrupts the phosphorylation site at Ser 16 of PLN, leading to impaired SERCA2a activity, abnormal calcium handling, and arrhythmia(2). Current therapy strategies are limited to symptom management and prevention of sudden cardiac death highlighting an urgent need for novel therapeutic strategies. The purpose of this study is to evaluate a novel Adeno-associated virus (AAV)-mediated treatment over-expressing the wild-type (WT) PLN, and to investigate how over-expression of the WT gene impacts cardiac function in a humanized PLN-R14del mouse model. Methods Three-month-old heterozygous (HET) hPLN-R14del mice were injected with 1 x 10^12 vg/mouse dose of AAV9-wtPLN vector, or saline control. Five weeks post-treatment, efficacy was assessed using an in-vivo stress induced arrhythmia protocol. A baseline ECG was recorded for 5 minutes, followed by a simultaneous intraperitoneal (IP) injection of epinephrine (2mg/Kg) and caffeine (120 mg/Kg), with an additional 10-15 minutes of post-injection ECG recording. The mice were subsequently sacrificed, and their hearts and other organs were collected for downstream analysis to determine the effectiveness of the AAV-mediated therapy. The ECGs were analyzed using the beat classifier module in LabChart8 to quantify the percentage of arrhythmic beats, while viral genome and PLN gene expression were assessed by qPCR and digital droplet PCR. Results Arrhythmia could be efficiently induced by the simultaneous IP injection of epinephrine and caffeine in PLN-R14del HET mice. HET mice show significantly more arrhythmia compared to WT mice. The efficiency of the AAV-mediated treatment was determined by the viral genome expression. Treated mice show sufficient expression of the viral genome compared to saline injected mice. The effectiveness of the AAV9-hPLN vector to over-express the WT PLN was assessed in HET treated mice, resulting in a 15% increase in WT PLN gene expression compared to saline injected mice, along with a corresponding increase in PLN protein levels. Functionally, this resulted in a significant reduction in arrhythmia in AAV-treated mice compared to controls. Conclusion AAV-mediated gene therapy has demonstrated sufficient efficiency in promoting the over-expression of the target gene in a preclinical model for PLN-R14del cardiomyopathy. A 15% increase in WT-PLN led to a significant improvement of the arrhythmogenic phenotype of HET mice, suggesting that even a modest restoration of PLN expression can reduce disease-related arrhythmogenicity and serve as a potential therapeutic strategy.

The exact mechanism of postbariatric hypoglycemia (PBH) is unclear, but β-cell mass expansion is hypothesized to play a role. We used [68Ga]Ga-NODAGA-exendin-4 positron emission tomography/computed tomography (PET/CT) to determine β-cell mass in individuals with and without PBH after Roux-en-Y gastric bypass surgery. β-Cell mass did not differ between individuals with and without PBH. Pancreas volume was lower in individuals with PBH compared with those without PBH. Our data argue against β-cell mass expansion to explain PBH after Roux-en-Y gastric bypass. Further study is required to understand PBH.

Abstract Gene therapy-based biological pacemakers have been proposed as an alternative to their hardware-based counterparts. In this context, short-term ectopic expression of the T-box transcription factor 18 (TBX18) in the ventricle reportedly generated potent short-term pacemaker function in various animal models. Here, we investigated the effect of adeno-associated virus (AAV)-mediated long-term expression of TBX18, and compared the outcome to that of the pacemaker ion channel Hcn2. Our findings revealed that CMV-driven ectopic TBX18 expression in mouse hearts led to severe cardiac fibrosis. At lower, non-fibrogenic levels, TBX18 maintained its transcriptional function but failed to induce pacemaker phenotypes. TBX18-expressing cells showed suppressed expression of key working myocardial genes, but the pacemaker gene program was not induced. Electrophysiological studies showed abnormal automaticity in TBX18-expressing cells, combined with prolonged repolarization and various current changes. However, no hyperpolarization-activated funny current was detected. In a complete AV-block rat model, AAV-mediated Hcn2 expression induced robust ectopic pacemaker activity in the presence of isoproterenol, whereas TBX18 expression neither generated such activities, nor augmented Hcn2-mediated pacing. In conclusion, at functional non-fibrogenic levels, TBX18 is neither sufficient nor necessary to induce pacemaker activity. In contrast, Hcn2 generates reliable pacing, making it a more viable candidate for biological pacemaker development.

Triglycerides and T cells play a key role in atherosclerosis, the leading cause of cardiovascular disease (CVD). Moderately elevated triglycerides have emerged as a causal risk factor, and T cells are a prominent component of atherosclerotic plaques. This cross-sectional study examined transcriptomic differences in T cells among patients with varying triglyceride levels via RNA sequencing. We analyzed CD4+ and CD8+ T cells from 49 participants, including those with primary (genetic) and secondary moderate hypertriglyceridemia, severe hypertriglyceridemia, and hypotriglyceridemia. Patients with primary moderate hypertriglyceridemia exhibited a proinflammatory transcriptomic profile, including increased interleukin-6 receptor (IL6R) expression, which is implicated in CVD risk. Similar patterns appeared in CD8+ T cells and, to a lesser extent, in secondary moderate hypertriglyceridemia patients. Conversely, transcriptomic differences were reversed in hypotriglyceridemia and absent in severe hypertriglyceridemia patients. These findings suggest that elevated triglycerides may contribute to CVD by promoting a proinflammatory transcriptomic profile in T cells.