A Gαq/11-IP3R1-ANO1 pacemaking axis underlies pressure-induced lymphatic chronotropy

Abstract

The frequency of spontaneous lymphatic contractions is exquisitely sensitive to changes in intraluminal pressure, enabling lymph transport to be matched to the filling state of the lymphatic capillary network. The mechanistic basis of this mechanosensitive response was investigated using ex vivo contraction assays of mouse popliteal collecting vessels. Based on electrophysiological and transcriptional similarities between lymphatic muscle cells (LMCs) and arterial smooth muscle, we hypothesized that pressure-dependent chronotropy shares a parallel signaling process with pressure-induced arterial depolarization/constriction. Thus, we probed two potential mechanisms: 1) pressure-induced activation of mechanosensitive cation channels and 2) mechano-activation of GNAQ/GNA11-coupled G-protein receptors (GPCRs) that would generate second messengers to activate those channels. Contraction assays were combined with scRNAseq analysis of the respective targets and use of transgenic mice. Our findings rule out significant roles for the mechanosensitive channels implicated in arterial myogenic constriction as well as channels implicated in ionic pacemaking of other tissues. In contrast, smooth-muscle specific knock out or inhibition of ANO1, IP3R1 or GNAQ/GNA11 significantly abrogated pressure-induced chronotropy. These results support a scheme whereby mechano-activation of one or more GNAQ/GNA11-coupled GPCRs generates IP3, inducing sarcoplasmic reticulum Ca2+ release through IP3R1, which drives depolarization through the activation of ANO1 Cl- channels. The major GPCRs expressed in LMCs were identified and ranked by scRNAseq analysis but individual inhibition or knock out the top 7 candidates failed to significantly impair pressure-induced chronotropy. Our results implicate one or more GNAQ/GNA11-coupled GPCRs in mediating this mechanosensitive process, but the specific GPCRs remain to be identified.