Analyzing Remodeling of Cardiac Tissue: A Comprehensive Approach Based on Confocal Microscopy and 3D Reconstructions

Abstract

Microstructural characterization of cardiac tissue and its remodeling in disease is a crucial step in many basic research projects. We present a comprehensive approach for three-dimensional characterization of cardiac tissue at the submicrometer scale. We developed a compression-free mounting method as well as labeling and imaging protocols that facilitate acquisition of three-dimensional image stacks with scanning confocal microscopy. We evaluated the approach with normal and infarcted ventricular tissue. We used the acquired image stacks for segmentation, quantitative analysis and visualization of important tissue components. In contrast to conventional mounting, compression-free mounting preserved cell shapes, capillary lumens and extracellular laminas. Furthermore, the new approach and imaging protocols resulted in high signal-to-noise ratios at depths up to 60 μm. This allowed extensive analyses revealing major differences in volume fractions and distribution of cardiomyocytes, blood vessels, fibroblasts, myofibroblasts and extracellular space in control versus infarct border zone. Our results show that the developed approach yields comprehensive data on microstructure of cardiac tissue and its remodeling in disease. In contrast to other approaches, it allows quantitative assessment of all major tissue components. Furthermore, we suggest that the approach will provide important data for physiological models of cardiac tissue at the submicrometer scale.

Remodeling of the transverse tubular system after myocardial infarction in rabbit correlates with local fibrosis: A potential role of biomechanics

“…T-system remodeling in failing rodent hearts has been studied extensively, but t-system components as found in human heart failure or in a porcine infarct model (Pinali et al, 2017) have not been identified. In contrast, images of remodeled t-tubules published by others (Driesen et al, 2007) and us (Seidel et al, 2016), including the present study, indicate that t-system remodeling in rabbit resembles the human phenotype. Driesen et al described vacuole-like structures in cardiomyocytes adjacent to the infarct border in rabbit and suggested that “the vacuoles represent plasma membrane invaginations and/or dilatations of t-tubular structures.” Based on our three-dimensional reconstructions (Fig.…”

“…We used 12 male New Zealand White rabbits of 2.5 to 3kg. In 6 randomly selected animals, left-ventricular MI was created by ligation of the left circumflex artery as previously described (Hu et al, 2010; Seidel et al, 2016). Successful ligation was confirmed by ST elevation in ECG recordings and epicardial demarcation of the infarcted area.…”

“…After immersion in 30% sucrose solution, slices of 80μm thickness were obtained by cryosectioning in a depth of 0.4 to 1.6mm below the epicardium as previously described (Seidel et al, 2016). Immediately after sectioning, and after each incubation step during staining, slices were rinsed in PBS.…”

“…A 10% overlap of tiles allowed automated registration and stitching using the Leica Application Suite X software package (versions: 1.1.0.12420 and 2.0.2.15022). We also acquired three-dimensional image stacks as described previously (Seidel et al, 2016). …”

“…Wheat germ agglutinin (WGA) binds to glycoproteins located in cell membranes and the extracellular matrix (Emde et al, 2014; Seidel et al, 2016). In rabbit and other species, it also binds to membranes of the t-system.…”