Heart gap junction preparations reveal hemiplaques by atomic force microscopy

Current models of gap junctions suggest two membranes with aligned hemichannels. These structures connect adjacent cells' cytoplasms. Prior research has used freeze-fracture and electron microscopy to study these junctions. However, the exact structure of hemiplaques remains unclear. This gap motivated the use of atomic force microscopy (AFM) in aqueous media. No prior work had resolved the native thickness of cardiac gap junctions. The role of trypsin in modifying junctional structures was already known. This study aimed to clarify the structural dynamics of gap junctions in heart tissue.

The aim was to investigate the structural properties of heart gap junctions using atomic force microscopy. Researchers sought to determine the thickness of native junctions and their response to trypsin treatment. They also aimed to identify and characterize novel structures observed in the AFM images. These structures were hypothesized to be hemiplaques. The study focused on structural changes induced by trypsin. The motivation was to better understand the formation and function of gap junctions. Prior methods had limitations in resolving fine structural details. This approach allowed for high-resolution imaging in physiological conditions.

Atomic force microscopy was used to image isolated rat heart gap junctions in aqueous media. The thickness of native junctions was measured before and after trypsin treatment. Long-range hexagonal packing was analyzed using AFM imaging. Center-to-center spacing of subunits was recorded. Freeze-fracture and negative-stain electron microscopy were used for comparison. The presence of hemiplaques was confirmed by their distinct thickness. AFM imaging was repeated under controlled conditions to rule out artifacts. The study combined structural analysis with biochemical treatment effects.

Native cardiac gap junctions had a thickness of 25 ± 0.6 nm. Trypsin treatment reduced this to 17 nm, consistent with cytoplasmic component removal. Subunits showed center-to-center spacing of 9-10 nm. Long-range hexagonal packing was observed in AFM images. Hemiplaques were identified with a thickness of 9-11 nm. These structures also showed hexagonal packing and 9-10 nm spacing. Trypsin further reduced hemiplaque thickness to 6-9 nm. The findings suggest hemiplaques are distinct from standard gap junctions.

The study confirms that gap junctions in heart tissue have a defined structural organization. Hemiplaques appear to be a distinct structure with similar packing but reduced thickness. Their presence was not an artifact of AFM imaging. The role of trypsin in modifying junctional components was validated. Hemiplaques may represent an intermediary in gap junction formation. The study does not assign essentiality to hemiplaques in junctional function. Their origin remains unclear, possibly linked to isolation procedures. Further research is needed to determine their functional significance.