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

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), 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 Ca(2+) transients; and depolarization-induced propagated contractions. We employed inducible Cre (iCre) mouse models routinely used to target these specific cell populations including: c-kitCreER(T2) to target ICLC; PdgfrβCreER(T2) to target pericytes; PdgfrαCreER(™) to target CD34(+) adventitial fibroblast-like cells or ICLC; and Myh11CreER(T2) to target LMCs. These specific inducible Cre lines were crossed to the fluorescent reporter ROSA26mT/mG, the genetically encoded Ca(2+) sensor GCaMP6f, and the light-activated cation channel rhodopsin2 (ChR2). c-KitCreER(T2) labeled both a sparse population of LECs and round adventitial cells that responded to the mast cell activator compound 48–80. PdgfrβCreER(T2) drove recombination in both adventitial cells and LMCs, limiting its power to discriminate a pericyte specific population. PdgfrαCreER(™) labeled a large population of interconnected, oak leaf-shaped cells primarily along the adventitial surface of the vessel. Titrated induction of the smooth muscle-specific Myh11CreER(T2) revealed a LMC population with heterogeneous morphology. Only LMCs consistently, but heterogeneously, displayed spontaneous Ca(2+) 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 by Myh11CreER(T2), but not PdgfrαCreER(™) or c-KitCreER(T2), resulted in a propagated contraction. These findings support the conclusion that LMCs, or a subset of LMCs, are responsible for mouse cLV pacemaking.