Unveiling the native architecture of adult cardiac tissue using the 3D-NaissI method

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Abstract Accurately imaging adult cardiac tissue in its native state is essential for regenerative medicine and understanding heart disease. Current fluorescence methods encounter challenges with tissue fixation. Here, we introduce the 3D-NaissI (3D-Native Tissue Imaging) method, enabling rapid, cost-effective imaging of fresh cardiac tissue samples in their closest native state, that we also extended to other tissues. We validated 3D-NaissI’s efficacy in preserving cardiac tissue integrity using small biopsies under hypothermic conditions in phosphate-buffered saline, offering unparalleled resolution in confocal microscopy for imaging fluorescent-small molecules/-antibodies. Compared to conventional histology, 3D-NaissI preserves cardiac tissue architecture and native protein epitopes, facilitating the use of a wide range of commercial antibodies without unmasking strategies. We successfully identified specific cardiac protein expression patterns in cardiomyocytes (CMs) from rodents and humans, including for the first time ACE2 localization in the lateral membrane/T-Tubules and SGTL2 in the sarcoplasmic reticulum. These findings shed light on COVID-19-related cardiac complications and suggest novel explanations for iSGLT2 therapeutic benefits in HFpEF patients. Additionally, we challenge the notion of "connexin-43 lateralization” in heart pathology, suggesting it may be an artifact of cardiac fixation, as 3D-NaissI clearly revealed native connexin-43 expression at the lateral membrane of healthy CMs. We also discovered previously undocumented periodic ring-like 3D structures formed by pericytes covering CMs’ lateral surfaces. These structures, positive for laminin-2, delineate a specific spatial architecture of laminin-2 receptors at the CM surface, highlighting the pivotal role of pericytes in CM function. Lastly, 3D-NaissI facilitates mapping native human protein expression in fresh cardiac autopsies, providing insights into both pathological and non-pathological contexts. Hence, 3D-NaissI offers unparalleled insights into native cardiac tissue biology and promises to advance our understanding of physiology and pathophysiology, surpassing standard histology in resolution and accuracy.

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