Discerning the role of IP3R1 activation by Gq/G11 signaling in murine lymphatic collecting vessel pacemaking

Abstract

Collecting lymphatic vessels (cLVs) exhibit spontaneous contractions with a pressure‐dependent frequency that increases with elevations in intraluminal pressure. Our recent work posits a putative role for lymphatic muscle cells (LMCs) as the pacemaker cell that regulates this pressure‐dependent phenomenon through activation of the Ca2+‐activated chloride channel anoctamin 1 (Ano1) in an IP3R1‐dependent process. However, the specific mechanisms underlying IP3R1 activation and SR Ca2+ release in lymphatic muscle cells is unknown. We tested the hypothesis that cLV pressure‐dependent pacemaking by the IP3R1‐Ano1 axis is downstream of the canonical Gq/G11‐PLC‐β signaling pathway. To this end, we crossed mice with the smooth muscle‐specific tamoxifen‐inducible Cre, MYH11CreERT2, to the fluorescent reporter ROSAmT/mG and sorted GFP+ LMCs from an isolated murine inguinal‐axillary (IngAx) lymphatic cLVs. Murine lymphatic muscle cell expression of Gq and G11 were assessed via RT‐PCR. We crossed MYH11CreERT2 mice with IP3R1fl/fl or G11KO/KO‐Gqfl/fl mice to achieve IP3R1ismKO (MYH11CreERT2‐IP3R1fl/fl) and double G11KO‐GqismKO (MYH11CreERT2‐ G11KO/KO‐Gqfl/fl) mice. We tested the function of the IngAx cLVs isolated from these mice, as well as their respective heterozygous WT/KO and fl/fl controls, using isolated vessel isobaric myography. Ca2+ imaging using IP3R1ismKO‐GCaMP6f mice, and membrane potential recordings using sharp electrodes. IngAx cLVs from G11KO‐GqismKO mice displayed a significant reduction in contraction frequency and vessel tone, however a pressure‐dependent increase in frequency was observed. Similarly, IngAx cLVs isolated from IP3R1ismKO mice had significantly lower vessel tone, a significant reduction in contraction frequency at all pressures tested, and a significantly higher contraction amplitude. Critically, contraction frequency in IngAx cLVs isolated from IP3R1ismKO mice did not increase with pressure in contrast to IngAx cLVs isolated from G11KO‐GqismKO mice and wildtype mice. In wildtype (WT) MYH11CreERT2‐GCaMP6f controls, Ca2+ puffs and/or Ca2+ waves were observed in the majority of LMCs during the diastolic period, intervening the bright global Ca2+ flashes indicative of Ca2+ entry through L‐type Ca2+ channels during the LMC action potential (AP). These local diastolic Ca2+ transients (CaTs) persisted in the presence of nifedipine, and the frequency and spatial spread of CaTs increased with pressure. Diastolic CaTs were largely absent in LMCs of IP3R1ismKO mice, and their diastolic depolarization recapitulated the lower rate observed in previous studies of Ano1smKO cLVs. However, APs recorded in IP3R1ismKO significantly increased AP plateau duration that corresponds with an increased Ca2+ flash duration. We conclude that the Gq/G11 dependent signaling is involved in pressure‐dependent pacemaking in murine cLVs, particularly at low pressure, but their deletion is insufficient to reproduce the IP3R1ismKO phenotype.