Cellular characterization of the mouse collecting lymphatic vessels reveals that lymphatic muscle cells are the innate pacemaker cells

Collecting lymphatic vessels (cLVs) exhibit spontaneous contractions with a pressure-dependent frequency, but the identity of the lymphatic pacemaker cell is still debated. Here we combined immunofluorescence and scRNAseq analyses with electrophysiological methods to examine the cellular constituents of the mouse cLV wall and assess whether any cell type exhibited morphological and functional processes characteristic of pacemaker cells. We employed inducible Cre (iCre) mouse models to target specific cell populations including c-kitCreER^T2to target interstitial cells of Cajal like cells;PdgfrβCreER^T2to target pericyte-like cells;PdgfrαCreER^TMto target CD34⁺adventitial cells; andMyh11CreER^T2to target lymphatic muscle cells (LMCs) directly. These inducible Cre lines were crossed to the fluorescent reporter ROSA26mT/mG, the genetically encoded Ca²⁺sensor GCaMP6f, and the light-activated cation channel rhodopsin2 (ChR2). Only LMCs consistently, but heterogeneously, displayed spontaneous Ca²⁺events during the diastolic period of the contraction cycle, and whose frequency was modulated in a pressure-dependent manner. Further, optogenetic depolarization with ChR2 only induced propagated contractions in LMCs. Membrane potential recordings in LMCs demonstrated that the rate of diastolic depolarization significantly correlated with contraction frequency. These findings support the conclusion that LMCs, or a subset of LMCs, are responsible for mouse cLV pacemaking.