Fibrose in het hart tijdens hartfalen: studie van heterogeniteit en reversibiliteit

Cardiac fibrosis (CF) is characterized by an excess of fibrillar collagen deposition in the extracellular matrix (ECM) and/or reduced degradation of the matrix in the heart. It accompanies almost all cardiac diseases and predicts poor prognosis. In heart failure (HF), it results in ventricular stiffening, impaired pump function, and arrhythmogenic remodelling, yet effective anti-fibrotic therapies remain limited. A key barrier is that cardiac fibroblasts (FB), the main effector cells, are highly heterogeneous across myocardial niches, disease aetiologies, and local microenvironmental cues, making broad inhibition of canonical pro-fibrotic pathways potentially harmful to homeostatic or reparative functions. Lineage studies further indicate that most activated FB derive from resident FB, suggesting that much of this heterogeneity reflects context-specific local cues. This thesis therefore examines FB heterogeneity in human HF to identify disease-relevant pathways that could be targeted to modulate (and potentially reverse) maladaptive fibrosis. These objectives were addressed primarily using single-nucleus RNA sequencing (snRNA-seq) integrated with advanced computational analyses, complemented by targeted in vitro perturbation experiments and validation. In the first study (Chapter 3), we mapped the FB landscape in human non-failing and failing myocardium due to ischemic and dilated cardiomyopathies (ICM and DCM respectively). We demonstrated that location and aetiology are major determinants of FB activation and heterogeneity. Three principal resident FB states were defined in non-failing hearts: spatially restricted interstitial (ECM-enriched) and perivascular (ECM-processing) populations, plus an immune-modulatory FB. With HF, distinct activated states emerged from these resident states, sharing a core activation program (e.g., COL1A1/POSTN/FAP) yet maintaining niche-linked features: interstitial FB largely remained in situ and aligned with interstitial fibrosis, whereas perivascular FB showed capacity to infiltrate scar regions, consistent with roles in scar remodelling. For these states, we identified common and unique activation ligands and transcription factors that facilitate this activation process. Importantly, disease-context signatures were identified: DCM fibroblasts showed prominent, cell-type specific circadian transcriptional disruption, while ICM scars contained specialized FB programs including senescence-associated and matrifibrocyte-like signatures, highlighting that scar versus non-scar fibrosis involves partially transcriptionally distinct FB states. We also identified non-scar signature related to matrix processing function. Together, MS1 argues that FB activation cannot be reduced to a single "TGF-β-driven" trajectory, but instead reflects microenvironmental context and divergent state transitions with translational relevance for precision anti-fibrotic targeting. In the second study (Chapter 4), we build on the outcomes of the 1st study and experimentally assessed one of the candidates identified. We focused on oxidative stress biology in human HF and identified an FB-selective vulnerability centred on MGST1 (microsomal glutathione S-transferase 1). Interrogation of snRNA-seq data showed that, across cardiac cell types, FB displayed the strongest downregulation of oxidative stress-modulating genes in HF; MGST1 was uniquely enriched in FB, decreased in failing hearts, and inversely correlated with elevated tissue ROS. In primary human FB, TGF-β1 driven activation reduced MGST1, linking MGST1 loss to the activation program. Functionally, MGST1 knockdown modestly shifted phenotype at baseline (e.g., increased periostin without a full collagen-induction program) but reduced stress resilience under external oxidative challenge: MGST1-deficient FB exhibited higher ROS accumulation, increased susceptibility to oxidative damage and lipid peroxidation, and transcriptional patterns consistent with reduced ferroptosis defence and altered iron handling. These results support a deleterious feedback model in which FB MGST1 loss amplifies oxidative stress and remodelling, identifying restoration of endogenous FB antioxidant capacity as a potential disease-modifying avenue. Collectively, the thesis reframes cardiac fibrosis in HF as an emergent property of spatially organized, aetiology-dependent FB states coupled to stress-response remodelling. Our work highlights FB activation in HF exhibit shared and conserved signatures across aetiologies, but also unique pathways that can be exploited for targeted interventions: modulating state transitions or microenvironmental ligands, prioritizing strategies that spare necessary reparative/scar-stabilizing programs, and augmenting endogenous antioxidant defences rather than relying on broadly acting systemic inhibitors. This integrated human-tissue approach provides both a conceptual framework and a resource for developing more precise anti-fibrotic therapies and biomarkers in HF.