Characterization of the cellular components of mouse collecting lymphatic vessels reveals that lymphatic muscle cells are the innate pacemaker cells regulating lymphatic contractions
Authors: Zawieja S.D. , Pea G.A. , Broyhill S.E. , Patro A. , Bromert K.H. , Norton C. E. , Kim H. J. , Sivasankaran S. K. , Li M. , Castorena-Gonzalez J.A. , Drumm B.T. , Davis M.J. 
Published: August 2023

Collecting 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) or the lymphatic muscle (LMCs) cells themselves. 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: a continuous if not contiguous network integrated into the electrical syncytium; 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 pericyte-like cells;PdgfrαCreERTMto target CD34+adventitial cells and ICLC; andMyh11CreERT2to target LMCs directly. 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. Of these cells, 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 under control ofMyh11CreERT2, but notPdgfrαCreERTMor c-KitCreERT2, resulted in propagated contractions upon photo-stimulation. 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.

Impact

The 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 PDGFRa+while vessel-associated cKit+cells are mast cells; and neither cell type demonstrated pacemaking capability.Myh11CreERT2identified lymphatic muscle cells that exhibited pacemaker behaviors such as pressure-dependent depolarization and calcium events during diastole and propagated contraction waves induced by focal, optical stimulation of channel-rhodopsin2.