Lymphatic muscle cells are the innate pacemaker cells regulating mouse lymphatic collecting vessel contractions

Abstract

AbstractCollecting lymphatic vessels (cLVs) exhibit spontaneous contractions with a pressure-dependent frequency, but the identity of the lymphatic pacemaker cell is still debated. By analogy to pacemakers in the GI and lower urinary tracts, proposed cLV pacemaker cells include interstitial cells of Cajal like cells (ICLC), pericytes, as well as the lymphatic muscle (LMCs) cells themselves. Here we tested the extent to which these cell types are invested into the mouse cLV wall and if any cell type exhibited morphological and functional processes characteristic of pacemaker cells: a contiguous network; spontaneous Ca2+transients; and depolarization-induced propagated contractions. We employed inducible Cre (iCre) mouse models routinely used to target these specific cell populations including: c-kitCreERT2to target ICLC;PdgfrβCreERT2to target pericytes;PdgfrαCreERTMto target CD34+adventitial fibroblast-like cells or ICLC; andMyh11CreERT2to target LMCs. These specific 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). c-KitCreERT2labeled both a sparse population of LECs and round adventitial cells that responded to the mast cell activator compound 48-80.PdgfrβCreERT2drove recombination in both adventitial cells and LMCs, limiting its power to discriminate a pericyte specific population.PdgfrαCreERTMlabeled a large population of interconnected, oak leaf-shaped cells primarily along the adventitial surface of the vessel. Titrated induction of the smooth muscle-specificMyh11CreERT2revealed a LMC population with heterogeneous morphology. 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. Optogenetic depolarization through the expression of ChR2 byMyh11CreERT2, but notPdgfrαCreERTMor c-KitCreERT2, resulted in a propagated contraction. These findings support the conclusion that LMCs, or a subset of LMCs, are responsible for mouse cLV pacemaking.ImpactThe presence and functionality of proposed pacemaker cells in collecting lymphatic vessels was tested with various mouse Cre models to drive expression of a recombination reporter ROSA26mT/mG, a genetically encoded Ca2+sensor GCaMP6f, or the optogenetic tool channel-rhodopsin2. Lymphatic CD34+adventitial cells co-express PDGFRΑ+while cKit+cells are mast cells; and neither cell type demonstrated pacemaking functionality.Myh11CreERT2identified lymphatic muscle cells which exhibited pacemaker behaviors such as pressure-dependent calcium events during diastole and propagated contraction induced by optical stimulation of channel-rhodopsin2.