Correlative and Live-Cell Imaging of S100B-Promoter-Associated Interstitial Cells from the Cardiac Sinoatrial Node and Valves

Cardiac pacemaker and valve cells operate within a heterogeneous interstitial environment, but the identities and observable behaviors of many constituent cells remain incompletely defined. We integrated complementary imaging observations from a single experimental platform into an exploratory analysis of cultured cells isolated from the adult mouse sinoatrial node (SAN) and atrioventricular valves of S100B-EGFP reporter mice. Cellular morphology, endogenous NAD(P)H fluorescence, tetramethylrhodamine methyl ester (TMRM) fluorescence, correlative light and electron microscopy (CLEM), time-lapse imaging, transmission electron microscopy (TEM), and 5-ethynyl-2′-deoxyuridine (EdU) labeling were used to describe selected reporter-positive and reporter-negative cells. S100B-promoter-associated EGFP+ cells displayed heterogeneous forms, including cells with small bodies and long processes. In representative fields, EGFP+ cells showed greater NAD(P)H autofluorescence intensity and lower TMRM signal than adjacent EGFP− cells. CLEM related selected fluorescence phenotypes to ultrastructure in the same cells and documented mitochondrial, vesicular, nuclear, and membrane-associated profiles. One live sequence captured movement of a TMRM-positive, mitochondrion-like structure through a thin intercellular bridge toward an EGFP− cell, followed by bridge disassembly. Additional images showed mitochondrial and vesicular profiles near cell surfaces or in extracellularly situated regions, elongated NAD(P)H-bright structures, reporter-positive cytoplasmic fragments, nuclear size heterogeneity, a narrow connection between nuclear profiles, and unequal EdU labeling among nuclear fragments. These findings are descriptive and hypothesis-generating. They do not establish definitive cell identity, phenotype prevalence, active or selective secretion, tunneling-nanotube identity, mitochondrial metabolic competence, regulated nuclear remodeling, recipient-cell uptake, or physiological consequence. The study provides an integrated morphological framework and a set of candidate events for future quantitative investigation of S100B-associated cardiac interstitial-cell biology.