Katrin Streckfuss-Bömeke

Takotsubo Syndrome (TTS) is an acute, potentially life-threatening condition often complicated by ventricular dysfunction and arrhythmias. Atrial fibrillation (AF) is common in TTS patients and associated with increased mortality. However, the arrhythmic mechanisms, genetic variability underlying TTS, and personalised therapy remain unclear. We generated induced pluripotent stem cell-derived atrial and ventricular cardiomyocytes (iPSC-CMs) from TTS patients and provoked TTS-like events by isoprenaline (Iso) stimulation. Compared with controls, atrial TTS iPSC-CMs exhibited enhanced diastolic sarcoplasmic reticulum (SR) Ca²⁺ leak and increased arrhythmogenic potential following Iso exposure. Iso-induced stress further increased systolic Ca²⁺ transient amplitude and reduced phosphodiesterase 4 (PDE4) activity in TTS iPSC-CMs. Under baseline conditions, atrial TTS iPSC-CMs demonstrated unchanged Ca ²⁺ transient amplitude, reduced diastolic Ca ²⁺ , elevated SERCA2 activity with faster Ca²⁺ decay compared to controls, which could be explained by increased phosphorylation of PKA-specific phospholamban S16. In contrast, ventricular TTS-iPSC-CM showed increased L-Type Ca ²⁺ current density, but reduced Ca ²⁺ transient amplitude with increased resting Ca ²⁺ leak and reduced PDE4 activity post-Iso-exposure. In atrial TTS iPSC-CMs, the PDE4 activator MR-L2 restored PDE4 activity, normalised several defects in Ca²⁺ handling and prevented arrhythmias more efficiently than the β-blocker metoprolol. Genetic analyses identified variants in the Ca²⁺ handling genes AHNAK and CASQ2 in TTS. Using CRISPR/Cas9, we generated isogenic rescue and mutation-insertion lines, confirming a causal role of p.P5390T in catecholamine-induced arrhythmogenic phenotypes in atrial TTS iPSC-CMs. Our findings support a mechanistic link between genetic predisposition to an excessive catecholaminergic stress response, dysregulated PDE/cAMP signalling, and atrial arrhythmogenesis in TTS-CMs, identifying PDE4 as a potential therapeutic target in this critical condition.

Mutations in the splice-regulator RBM20 cause heart failure with reduced ejection fraction (HFrEF), typically manifesting as dilated cardiomyopathy (DCM). Mutations at position 634 in the RS-domain cause DCM with (R634L) or without (R634W) left ventricular non-compaction (LVNC). However, the mechanisms underlying phenotype variability and personalized therapy beyond HFrEF remain unclear. We generated induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM), 3D-cardiospheres and engineered myocardial tissues from patients with RBM20 mutations R634L (LVNC) or R634W (DCM). Using CRISPR/Cas9, we created isogenic rescue and mutation-insertion lines, identifying RBM20 mis-localization, splicing errors in TTN and RYR2, and sarcomere irregularities in both. DCM-CM showed increased resting Ca²⁺ leak and reduced Ca²⁺ transient amplitude, typical of HFrEF, and spatial disorganization of sarcoplasmic reticulum and mitochondria. In contrast, LVNC-CM exhibited elevated Ca²⁺ transient amplitude with faster kinetics, driven by elevated cAMP and mis-spliced, hyperactive CAMK2D, leading to PLN-hyperphosphorylation and increased metabolic respiration. Further, LVNC showed desmosomal derangement potentially from mis-splicing of Junction plakoglobin and reduced 3D cardiosphere compaction. Despite distinct mechanisms, contractile force was reduced in both. Isogenic controls confirm mutation causality. Drug intervention with verapamil partially improved selected abnormal Ca²⁺ handling and contractile phenotypes in LVNC- and DCM-CM, whereas the CAMK2D inhibitor AIP improved systolic Ca²⁺ handling predominantly in LVNC-CM. In conclusion, different amino acid substitutions at the same RBM20-residue induce opposing Ca²⁺-handling and structural phenotypes. While DCM features impaired Ca²⁺ handling, LVNC shows defective cell-cell coupling and activated Ca²⁺ handling and metabolism, yet insufficient to compensate for organ-level dysfunction. This supports personalized pharmacological therapies in early HF, and potential CRISPR/Cas9 repair for RBM20 cardiomyopathy.

Model systems that mimic human cardiac structure and function are essential for the development of novel diagnostics and effective treatments for cardiovascular diseases. While non‐human vertebrate models, from zebrafish to pig, remain vital to cardiovascular research, the translatability of findings to human patients is often limited. Therefore, animal experiments should be supplemented with human model systems, including human induced pluripotent stem cell‐derived cells, 3D engineered constructs, and last but not least, native tissue preparations and isolated primary cardiomyocytes. However, while human myocardium remains the gold standard, human heart tissue – and particularly tissue from control hearts–remains scarce, and its use in research is generally restricted to settings where tissue has been excised from diseased or failing hearts. While it is in principle possible to use tissue from rejected non‐failing donor hearts that cannot be transplanted, legal hurdles (e.g., in Germany) can restrict the use of non‐transplanted donor organs in research. Given the challenges associated with accessing and using human tissue in biomedical research, an integrated strategy towards combining non‐human vertebrate models, in silico models, and human tissue‐derived models is recommended, enhancing the chances of successful research and development, and helping bridge the gap between preclinical and clinical research.

Transcriptional activity perturbation holds promise for selectively modulating harmful transcriptional networks, but its therapeutic potential remains largely unexplored. We employed a network-based analysis of single-cell heart transcriptomes to identify transcription factor activities linked to pathological cardiomyocytes in vivo. This analysis revealed that transcriptional activity of Krüppel-like factor 15 (KLF15) exhibited the most significant change in pathological cardiomyocytes, characterized by less effective repression of disease-associated genes in stressed hearts, which correlated with reduced KLF15 expression. To restore KLF15 activity, we utilized CRISPR/nuclease-dead (d)Cas9-based transcriptional enhancement (CRISPRa) in cardiomyocytes, which effectively abolished fetal reprogramming by simultaneously suppressing pathological gene expression and restoring metabolic homeostasis under sustained stress conditions. Furthermore, we identified a novel cell-nonautonomous anti-fibrotic effect mediated by cardiomyocyte-fibroblast crosstalk, and revealed the contribution of KLF15-dependent Alpha-2-glycoprotein 1, zinc-binding (AZGP1) regulation in this process. We also elucidated the upstream mechanisms of KLF15 regulation, highlighting its role as a cell-specific downstream target of the broad TGF-β canonical signaling pathway, along with its downstream-dependent mechanisms in human cardiomyocytes. Finally, to enhance the therapeutic potential of this approach, we engineered and validated an adeno-associated viral (AAV) vector with a small CRISPRa system for endogenous regulation in human cardiomyocytes suitable for clinical applications. Overall, we elucidated a regulatory circuit involving TGF-β, KLF15, and AZGP1, which coordinates critical pathological responses through cellular crosstalk between cardiomyocytes and fibroblasts. Importantly, we demonstrated the efficacy of CRISPRa as an epigenetic intervention restoring a critical transcriptional function disrupted in non-genetic heart failure. This approach provides a promising blueprint for future adaptation targeting additional non-hereditary pathologies.

Model systems that mimic human cardiac structure and function are essential for the development of novel diagnostics and effective treatments for cardiovascular diseases. While non-human vertebrate models, from zebrafish to pig, remain vital to cardiovascular research, the translatability of findings to human patients is often limited. Therefore, animal experiments should be supplemented with human model systems, including human induced pluripotent stem cell-derived cells, 3D engineered constructs, and last but not least, native tissue preparations and isolated primary cardiomyocytes. However, while human myocardium remains the gold standard, human heart tissue – and particularly tissue from control hearts – remains scarce, and its use in research is generally restricted to settings where tissue has been excised from diseased or failing hearts. While it is in principle possible to use tissue from rejected non-failing donor hearts that cannot be transplanted, legal hurdles (for example in Germany) can restrict the use of non-transplanted donor organs in research. Given the challenges associated with accessing and using human tissue in biomedical research, an integrated strategy towards combining non-human vertebrate models, in silico models, and human tissue-derived models is recommended, enhancing the chances of successful research and development, and helping bridge the gap between preclinical and clinical research.

Zusammenfassung Herzrhythmusstörungen tragen wesentlich zur Morbidität und Mortalität bei Patienten mit Herz-Kreislauf-Erkrankungen bei. Neben den häufig erworbenen Ursachen stellen auch hereditäre Ionenkanalerkrankungen wie das Brugada-Syndrom, das Long‑/Short-QT-Syndrom und die katecholaminerge polymorphe ventrikuläre Tachykardie (CPVT) wichtige Entitäten mit erhöhtem Risiko für ventrikuläre Arrhythmien dar. Obwohl Betablocker, Ionenkanalmodulatoren und implantierbare Kardioverter-Defibrillatoren (ICD) zur Verfügung stehen und deren Indikation zunehmend auch auf genetischen Analysen basiert, erschweren Varianten unklarer Signifikanz (VUS) die Risikostratifizierung erheblich. Atriale Tachyarrhythmien, insbesondere das Vorhofflimmern, können eine oft reversible Arrhythmie-induzierte Kardiomyopathie (AIC) verursachen. Pathophysiologisch nimmt die Ca²⁺/Calmodulin-abhängige Proteinkinase II (CaMKII) eine wichtige Rolle bei atrialen und ventrikulären Arrhythmien ein. Sie kann durch verschiedene pathologische Auslöser aktiviert werden, wie hohe Herzfrequenz, neuroendokrine Aktivierung und oxidativer Stress, letzterer häufig als Resultat einer mechanoenergetischen Entkopplung. Die Entwicklung wirksamer Therapien für arrhythmieassoziierte kardiovaskuläre Erkrankungen erfordert aufgrund genetischer Heterogenität und systemischer Komorbiditäten neben tierexperimentellen Ansätzen auch den Einsatz humaner Modellsysteme. So ermöglichen patientenspezifische kardiale Stammzellmodelle die Klassifikation von VUS und personalisierte Arzneimitteltests. Die Integration neuer methodischer Ansätze in die bestehenden tierexperimentellen Ansätze eröffnet damit den Weg zu einer funktionell validierten Präzisionsmedizin und könnte die Therapie beider Krankheitsbilder grundlegend transformieren.

Cis- regulatory elements (CREs) are noncoding DNA regions regulating cell-type-specific gene expression programs by interacting with distal gene promoters. Here, we aim to decode the function and spatial organization of CRE-promoter interactions in human cardiomyocytes. We analyzed the epigenome and chromatin interactions of human male atrial, ventricular, and failing cardiomyocytes. Atrial and ventricular cardiomyocytes harbored chamber-specific CRE-promoter interactions modulating gene expression as confirmed by functional epigenetic silencing. These CRE-promoter interactions explain the distinct contribution of non-coding genetic variants to atrial and ventricular diseases, such as dilated cardiomyopathy and arrhythmias. We dissected the prototypic KCNJ2 locus, encoding a potassium channel associated with ventricular arrhythmia susceptibility. Functional epigenetic silencing confirmed that CREs, harboring QT-duration-associated genetic risk factors, modulate KCNJ2 gene expression levels, alter KCNJ2-dependent channel currents, and affect cardiomyocyte repolarization. The presented human CM-specific chromatin interaction analysis provides key insights into regulatory mechanisms and aids in interpreting genetic risk factors.

BACKGROUND: Mitochondrial calcium (Ca ²⁺ ) is a key regulator of cardiac energetics by stimulating the tricarboxylic acid cycle during elevated workload. Atrial fibrillation (AF) is associated with a reduction in cytosolic Ca ²⁺ transient amplitude, but its effect on mitochondrial Ca ²⁺ handling and cellular redox state has not been explored. METHODS: Cardiac myocytes isolated from patient-derived right atrial biopsies were subjected to workload transitions using patch-clamp stimulation and β-adrenergic stimulation (isoproterenol). In conjunction, nicotinamide adenine dinucleotide (phosphate)/flavin adenine dinucleotide (NAD[P]H/FAD) autofluorescence, cytosolic and mitochondrial [Ca ²⁺ ] were monitored using epifluorescence microscopy. Sarcoplasmic reticulum and mitochondria were imaged using electron microscopy and tomography and stimulated emission depletion microscopy. The effects of the mitochondrial Ca ²⁺ uptake enhancer ezetimibe on proarrhythmic activity in atrial myocytes and on AF burden in patients were investigated. RESULTS: Mitochondrial Ca ²⁺ accumulation during increased workload was blunted in AF, and was associated with impaired regeneration of nicotinamide adenine dinucleotide and flavin adenine dinucleotide. Nanoscale imaging revealed spatial disorganization of sarcoplasmic reticulum and mitochondria, associated with microtubule destabilization. This was confirmed in human induced pluripotent stem cell–derived cardiac myocytes, where treatment with the microtubule destabilizer nocodazole displaced mitochondria and increased proarrhythmic Ca ²⁺ sparks, which were rescued by MitoTEMPO. Ezetimibe also reduced the occurrence of arrhythmogenic Ca ²⁺ release events both in AF myocytes and nocodazole-treated human induced pluripotent stem cell–derived cardiac myocytes. Retrospective patient analysis also revealed a reduced AF burden in patients on ezetimibe treatment. CONCLUSIONS: Mitochondrial Ca ²⁺ uptake and accumulation are impaired in atrial myocytes from patients with AF. The disturbed spatial association between sarcoplasmic reticulum and mitochondria driven by destabilized microtubules may underlie impaired Ca ²⁺ transfer in AF. Enhancing mitochondrial Ca ²⁺ uptake potentially protects against arrhythmogenic events.