Targeted Isolation and Functional Characterization of Periganglionic Cardiac Telocytes

Abstract Cardiac telocytes are specialized interstitial stromal cells implicated in tissue organization, intercellular communication, and cardiac remodeling. However, telocytes associated with the intrinsic cardiac nervous system remain poorly characterized, partly because of the lack of targeted isolation approaches. Here, we describe an optimized strategy for the isolation and functional characterization of telocyte-like cells from the intracardiac ganglionic region of the rat heart. Anatomically guided microdissection of the dorsal atrial ganglionic region was combined with sequential enzymatic dissociation and short-term primary culture. Isolated cells displayed characteristic telocyte-like morphology, including elongated cell bodies and moniliform telopodes with alternating podomers and podoms, and expressed the telocyte-associated markers CD34, PDGFRα, and vimentin. Immunofluorescence analysis further demonstrated elongated CD34-positive stromal cells within the connective tissue surrounding intracardiac ganglia. Functional competence was assessed using Fluo-4 AM-based intracellular Ca²⁺ imaging. Angiotensin II (1.4 µM) and caffeine (1.2 mM) significantly increased Ca²⁺-dependent fluorescence (P < 0.0001 for both), with representative recordings showing a sustained post-peak response following angiotensin II stimulation and a faster, predominantly transient response following caffeine stimulation. Collectively, these findings establish an anatomically targeted approach for isolating functionally responsive periganglionic cardiac telocyte-like cells and provide an experimental platform for investigating telocyte biology and neuro-stromal interactions within the intrinsic cardiac nervous system.