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

Abstract

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-kitCreERT2 to target interstitial cells of Cajal like cells; Pdgfr{beta}CreERT2to target pericyte-like cells; PdgfrCreERTMto target CD34+ adventitial cells; and Myh11CreERT2to 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. ImpactLymphatic muscle cells, but not CD34+ adventitial cells, exhibited pacemaker behaviors such as pressure-dependent depolarization, pressure-dependent calcium mobilization during diastole, and propagated contraction waves induced by focal, optogenetic depolarization via enforced channel- rhodopsin2.