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 microm. This allowed extensive analyzes revealing major differences in volume fractions and distribution of cardiomyocytes, blood vessels, fibroblasts, myofibroblasts and extracellular space in control vs. 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.

Abstract:

Bidomain simulations of the heart need validated parameters to produce realistic data. Therefore, it is nec- essary to develop methods to estimate reliable values for these parameters. We developed an approach to deliver such values by designing an in-silico model of intracellular electrical conduction based on confocal microscopic data of rabbit ventricular tissue. High resolution image data were used to determine the anisotropy of electrical conduc- tivity in the myocardium, which is highly dependent on the specific tissue geometry. Gap junction protein connexin43 and extracellular space were labeled with fluorescent dyes of different spectra. The myocytes were segmented and the gap junction density in-between myocytes was extracted. Assuming conductivities for intracellular liquid and gap junction resistance, a numerical field calculation was per- formed for three principal directions in order to extract in- tracellular conductivity tensors. We calculated 9 tensors by varying the assumed conductivities by ±50%. We esti- mated the intracellular conductivities for the three princi- pal directions σi,x = 0.0653 S/m, σi,y = 0.0042 S/m and σi,z = 0.0033 S/m, respectively. The estimated conductiv- ity values were realistic regarding the electrical anisotropy but need to be improved to fit other experimental data.