Electrical interaction between cardiomyocyte sheets separated by non-cardiomyocyte sheets in heterogeneous tissues

Electrical coupling among cardiomyocytes is essential for synchronized heart contractions and proper cardiac function. While cardiomyocytes are known to interact with non-cardiomyocytes, the specific mechanisms and timing of these interactions remain unclear. Prior research has shown that disruptions in these couplings can lead to arrhythmias and impaired heart function. However, the role of non-cardiomyocyte layers in modulating electrical communication between cardiomyocyte sheets has not been fully explored. This gap motivated the investigation into how the number of intervening non-cardiomyocyte layers affects the timing and completeness of electrical coupling. Understanding these interactions could provide insights into the design of engineered cardiac tissues. Current studies focus on the electrical behavior of layered cell sheets but lack detailed time-course analyses of heterocellular coupling. This paper addresses that limitation by examining the temporal dynamics of electrical interactions in a controlled three-dimensional setup.

The study aimed to investigate how non-cardiomyocyte layers influence the electrical coupling between cardiomyocyte sheets. Researchers focused on the time-dependent changes in electrical communication when cardiomyocyte sheets were separated by varying numbers of NIH3T3 fibroblast layers. The specific problem addressed was the need to understand how the number of intervening non-cardiomyocyte layers affects the speed and completeness of electrical coupling. This question is important because it could inform the design of tissue-engineered cardiac constructs. The motivation for the study stems from the observation that electrical coupling is critical for normal heart function. The researchers sought to determine whether the presence of non-cardiomyocyte layers could delay or block electrical conduction between cardiomyocyte sheets. The study also aimed to explore whether gap junctions formed at heterocellular junctions could explain the observed coupling patterns. By analyzing electrical interactions in a three-dimensional model, the researchers hoped to simulate conditions relevant to native or damaged heart tissue.

The researchers used a multiple-electrode extracellular recording system to analyze the three-dimensional time course of electrical interactions. They created layered cell sheets consisting of rat neonatal cardiomyocytes and NIH3T3 fibroblasts. The cardiomyocyte sheets were separated by one, two, or three layers of NIH3T3 cells. Electrical coupling was measured by tracking the time it took for electrical signals to propagate between the cardiomyocyte sheets. The study also included control groups using HeLa cell sheets and Ca(2+)-antagonist-treated cell sheets to assess conduction blockage. Immunocytological analysis was performed to detect the presence of gap junction proteins at heterocellular junctions. Dye transfer assays were used to confirm the formation of functional gap junctions between cardiomyocytes and NIH3T3 cells. The experimental setup allowed for precise timing measurements of electrical coupling events, providing a detailed temporal profile of the interactions.

The study found that cardiomyocyte sheets separated by a single-layer NIH3T3 cell sheet coupled electrically at 113 ± 28 minutes after layering. When separated by a double-layered NIH3T3 cell sheet, electrical coupling occurred at 287 ± 87 minutes. The time course of coupling with a single-layer NIH3T3 sheet was similar to that of a layered cardiomyocyte sheet. However, the double-layered NIH3T3 sheet caused a conduction delay, indicating incomplete electrical coupling. A triple-layered NIH3T3 sheet, a communication-defective HeLa cell sheet, or a LaCl(3)-treated cell sheet completely blocked electrical coupling. Immunocytological analysis showed the formation of gap junctions at heterocellular junctions between cardiomyocytes and NIH3T3 cells. Dye transfer assays confirmed the presence of functional gap junctions. These findings suggest that the number of intervening non-cardiomyocyte layers significantly affects the timing and completeness of electrical coupling between cardiomyocyte sheets.

The authors concluded that the number of intervening non-cardiomyocyte layers influences the timing and completeness of electrical coupling between cardiomyocyte sheets. Single-layer NIH3T3 sheets allowed for relatively rapid coupling, while double-layered sheets introduced conduction delays. Triple-layered sheets or communication-defective cell sheets completely blocked electrical coupling. The presence of gap junctions at heterocellular junctions supports the role of these structures in facilitating electrical communication. These findings may provide insights into the electrical conduction systems of native or damaged heart tissues. The study also suggests that engineered cardiac tissues could be designed with specific non-cardiomyocyte layers to modulate electrical coupling. The observed conduction delays and complete blocks indicate that the thickness of non-cardiomyocyte layers can significantly impact cardiac function. These results highlight the importance of considering heterocellular interactions in the design of tissue-engineered constructs.