1Universitätsklinikum Regensburg Klinik und Poliklinik für Innere Med. II, Kardiologie Regensburg, Deutschland
Introduction: Tachycardiomyopathy is triggered by tachycardia, implies reversible left ventricular systolic dysfunction and can potentially lead to heart failure. It entails a specific ultrastructural fingerprint, setting it apart from afterload- or ischemia-induced heart failure. However, a comprehensive, quantitative description of nuclear and cellular morphology in tachycardiomyopathy is still lacking. It could provide new insights into differences between reversible and non-reversible cardiomyopathies.
Study aim: That is why in this study we implemented an automated image analysis platform to extract several morphometric descriptors of nuclear and cellular composition. Additionally, we implemented quantitative parameters to describe cardiomyocyte disarray, which plays a vital role during pathophysiological remodeling of the heart muscle in other cardiomyopathies.
Methods: Tachycardiomyopathy was induced by permanent pacemakers in rabbits. During 30 days, heart frequency was gradually increased to finally 380bpm (T-CM, n=8). In the sham group (SHAM, n = 11) the pacemaker remained inactive. Left and right ventricular specimens were harvested and stained for Isolectin-B and DAPI. Twenty images per rabbit and ventricle were digitalized and loaded into Matlab. We applied custom software to extract 48 nuclear morphologic descriptors as well as parameters of nuclear orientation (Figure, G & H).
Results: In T-CM, left ventricular systolic function decreased (T-CM vs. SHAM, fractional shortening 20.5 vs. 36.8%, p<0.0001), which resulted in pleural and pericardial effusion. Analysis of nuclear DAPI staining showed increased nuclear size in T-CM (AUC = 0.6614, p = 0.0091, Figure, A, B & D). Our qualitative observation, that nuclear shape – mainly eccentricity – was more heterogeneous in T-CM, could not be confirmed, although a certain trend could be seen (AUC = 0.5905, p = 0.1336). Interestingly, the two groups differed in parameters of nuclear texture heterogeneity such as Contrast(AUC = 0.6359, p = 0.0242) and Energy (Figure, F). It is possible that these changes in nuclear texture - indicative of chromatin reorganization - yield the visual observation of more heterogeneously shaped nuclei. Finally, T-CM showed signs of decreased orientation order as a manifestation of cardiomyocyte disarray indicated by higher Rotation (Figure, C & E) and a trend towards a lower Orientation Order Parameter (AUC = 0.5900, p = 0.1356) and decreased Orientation Co-Occurrence (AUC = 0.5686, p = 0.2550).
Conclusions: Several structural changes of nuclear morphology could be observed and quantitatively described in tachycardiomyopathy by our novel software tool. This approach can be seen as a framework to be used in other cardiomyopathies to further characterize structural differences in reversible and non-reversible cardiomyopathies.
Analyses of nuclear morphology in tachycardiomyopathy (T-CM) in rabbits
(A) Original 4´,6-diammidino-2-phenylindole (DAPI) Image (B) Automatically segmented nuclear mask (C) Illustration of nuclear orientation using yellow arrows (D) Nuclear size in left ventricular specimens of SHAM and T-CM (E) Nuclear mean Rotation in left ventricular specimens of SHAM and T-CM (F) Nuclear GLCM derived parameter Energy in left ventricular specimens of SHAM and T-CM (G) Exemplary formulae of GLCM derived descriptor Contrast and Energy (H) formulae of the Orientation Order Parameter (OOP) and the Orientation Co-Occurrence Parameter (OCP)