Analysis of Location-Dependent Cardiomyocyte Branching

Cardiomyocytes branch and interconnect with one another, providing important redundancy for propagation of electro-chemical signals. Despite this, cardiomyocyte branching structure remains poorly understood. Herein, myocardium from spontaneously hypertensive rats (SHR) was imaged using extended volume confocal microscopy. Samples from untreated SHRs (\(n = 2\)) were compared with SHRs undergoing ACE inhibitor treatment (\(n = 2\)). From these image-stacks the cardiomyocyte network (center-lines and branches) were manually tracked. The frequency of cardiomyocyte branching was calculated and these branching frequencies were compared according to spatial position within a myocardial sheetlet. ACE inhibitor treatment resulted in significantly reduced total cardiomyocyte branching compared with untreated SHR myocardium at 24 mo (0.49 ± 0.04 vs 1.07 ± 0.15 branches per 100 \(\upmu \)m, \(P = 0.020\)). Cardiomyocytes on the sheetlet-surface branched more frequently within their respective cell-layer (0.59 ± 0.07 branches per 100 \(\upmu \)m) compared with cardiomyocytes in the sheetlet interior (0.29 ± 0.12 branches per 100 \(\upmu \)m). The cardiomyocytes in the sheetlet interior exhibited more frequent between-layer branching (0.56 ± 0.06 branches per 100 \(\upmu \)m) compared to sheetlet-surface cardiomyocytes (0.17 ± 0.03 branches per 100 \(\upmu \)m). The ratio of within-layer to between-layer branching was significantly greater at the surface layer compared with the interior layer (3.93 ± 1.06 vs 0.47 ± 0.16, \(P=0.018\)). This proof-of-concept study demonstrates an approach to measuring branching cardiomyocyte networks and shows the spatial heterogeneity of cardiomyocyte branching.