Investigating Cellular Morphology in Right Ventricular Myectomies from the Tetralogy of Fallot Patients

Objectives: The tetralogy of Fallot (TOF) patients are at risk for life-threatening arrhythmias which is believed to be due, in parts at least, to connective tissue remodeling. We aimed to establish an advanced 3D imaging and analysis protocol to investigate changes in nonmyocyte (NM) morphology which may accompany the increase in extracellular matrix in the right ventricular (RV) outflow tract (RVOT) of TOF patients.

Methods: Fresh pediatric RV and RVOT myectomies were cryoprotected with a sucrose-gradient, frozen in OCT compound, and sectioned to 100-µm thick slices. Immunofluorescence staining protocols labeling nuclei, cardiomyocyte (CM) membranes, NM cytoplasm, and gap junction protein connexin43 (Cx43) were optimized to increase antibody penetration, and refractive index matching throughout the sample was improved. RVOT slices from four younger unrepaired TOF patients (TOFy, age: 6–16 months), two older unrepaired TOF patients (TOFo, age: 3–11 years), four younger patients with atrial septal defect (ASDy, age: 10–20 months), and RV slices from two older healthy donor hearts (CTRLo, age: 3–8 years) were then imaged with confocal microscopy. We developed Python- and C++-based algorithms to quantify cellular composition, morphometric features of CM, NM, and their nuclei, and the extent of NM/Cx43 overlap (as a possible indicator of heterocellular coupling) in 3D space, taking into consideration the complex RVOT anatomy. We compared TOFy versus ASDy, TOFy versus TOFo, and TOFo versus CTRLo.

Result: Optimized sample preparation enabled an increased imaging depth of over 100 µm without tissue clearing. Nuclear segmentation and classification errors were low with 5.79 ± 0.78 and 1.81 ± 1.18%, respectively. Overall, intraindividual and intragroup variability was high. Compared with CTRLo, TOFo demonstrated lower NM nuclei density, larger nuclei, and larger size of individual NM. Compared with ASDy, TOFy showed higher CM nuclei density, and smaller overall NM volume and individual NM size. With increasing age, overall NM volume decreased while size of individual NM increased in TOF. In all four groups, 2 to 10% of Cx43 was colocalized with NM. Presence of cyanosis in tissue donors at the time of surgery showed no effect on any of the given parameters.

Conclusion: Our 3D fluorescence imaging and analysis algorithms allow the quantification of cellular and nuclear geometry of CM and NM in TOF myocardium irrespective of CM orientation, providing a reliable basis for future disease phenotype-specific assessment.

Publication History

Article published online:
21 February 2021