Standardized Processing of Explanted Hearts After Stereotactic Arrhythmia Radiotherapy (Bio-STAR): A STOPSTORM Framework and First Human Findings

Figures

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  Figure 1. Integration of electroanatomical mapping with CT imaging for STAR treatment planning. Electroanatomical mapping voltage map (A) displaying three distinct myocardial regions selected for analysis: minimally fibrotic, irradiated (MIFI, blue); fibrotic, irradiated (FIRI, red); and non-fibrotic, non-irradiated control region (NFNI, green). Electroanatomical mapping data are integrated in the pre-procedural CT (B) to define the target volume (C) and register it in the planning-CT for dose calculation of the stereotactic arrhythmia radioablation (D: STAR). The resulting registration enables the treatment planning system to calculate and visualize isodose distributions delivered during STAR, displayed as colour-coded isodose areas (see dose legend). The target areas (GTV, gross target volume; PTV, planning target volume) correspond to VT circuit origins (annotated by blue and red arrows).

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  Figure 2. Systematic dissection and standardized sampling strategy for explanted myocardium. Schematic depiction of myocardial regions selected for histology, molecular, and cellular assays following STAR treatment. Yellow indicates necessary and green optional samples, labelled according to origin: non-fibrotic, non-irradiated, minimally fibrotic, irradiated, and fibrotic, irradiated. Processing methods include fixation in glutaraldehyde for microscopy, snap freezing in liquid nitrogen (N2) for molecular assays, stabilization in RNAlater for RNA-tests, buffer preservation for cell-assays, fixation in 10% neutral-buffered formalin or 4% formaldehyde (FA) and paraffin embedding for histology or freezing in optimal cutting temperature compound (e.g. Tissue-Tek) for cryostat sectioning or immunofluorescence. For immunohistochemistry 4% paraformaldehyde solutions (with methanol stabilizer) are preferred over FA.

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  Figure 3. Histology of human myocardial regions at 5 months post-STAR stained with (haematoxylin-eosin, first two rows at 10/40× magnification) and Picrosirius red (third row). All regions showed fatty infiltration (40×) and fibrotic remodelling (red), with the FIRI area displaying the most severe changes of dense collagen deposition, isolated myocytes and fully acellular zones.

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  Figure 4. Exemplary immunofluorescence images showing subcellular localization of Cx43 (magenta) and ryanodine receptor (RyR, green) in isolated cardiomyocytes from each region (Patient #1). Scale bars = 10 µm.

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  Figure 5. Exploratory investigation of regional variation in protein expression in irradiated and fibrotic human myocardium from two patients. (A) Representative Western blot analysis of key cardiac proteins in samples from non-irradiated (NFNINFNI), minimally fibrotic irradiated, and fibrotic irradiated regions with high (FIRI-HR) and lower (FIRI-LR) degree of macroscopic fatty infiltration for both patients. GAPDH or MLC2v served as loading controls. Red arrowhead indicates re-expression of atrial natriuretic peptide and MLC2a in FIRI-HR. (B) Densitometric quantification of protein expression normalized to NFNI and respective loading controls. Expression of MLC2a and ANP was markedly increased in FIRI-HR samples, suggesting enhanced stress-related remodelling. Protein expression of CaV1.2, NaV1.5 and SERCA is increased in FIRI-HR, too, but only if normalized to MLC2v as cardiomyocyte marker which likely reflects the combination of cardiomyocyte loss and possible changes in channel expression per cardiomyocyte. Cx43 protein levels also exhibited regional variation, with significant differences observed depending on normalization strategy. Statistical analysis was performed using linear…

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  Figure 6. Exploratory functional assessment and mitochondrial content in isolated cardiomyocytes from distinct myocardial regions of two explanted human hearts. (A) Representative confocal microscopy images of isolated cardiomyocytes stained with MitoTracker (left), illustrating mitochondrial distribution in cells derived from non-irradiated (NFNI), minimally fibrotic irradiated, and fibrotic irradiated regions. Scale bars = 10 µm. Quantitative analysis of mitochondrial density (% area) across regions (right). (B) Representative traces for both patients of intracellular calcium transients recorded at 1 Hz electrical field stimulation. Despite pacing at 1 Hz, many cells responded only at 0.5 Hz, reflecting altered excitability. Summary of calcium handling kinetics including transient amplitude (F/F0), time to 50% peak (TF50), time to peak (TTP), and decay constant (Tau) in NFNI, MIFI, and FIRI cardiomyocytes at (C) 0.5 and 1 Hz (D). Minimally fibrotic irradiated and fibrotic irradiated cells exhibited altered calcium signalling during excitation contraction coupling as compared to NFNI cells, indicating heterogeneous calcium signalling characteristics among analysable cardiomyocytes from…