IP3R1 underlies diastolic ANO1 activation and pressure-dependent chronotropy in lymphatic collecting vessels

Abstract

Pressure-dependent chronotropy of murine lymphatic collecting vessels relies on the activation of the Ca2+-activated chloride channel encoded by Anoctamin 1 (Ano1) in lymphatic muscle cells. Genetic ablation or pharmacological inhibition of ANO1 results in a significant reduction in basal contraction frequency and essentially complete loss of pressure-dependent frequency modulation by decreasing the rate of the diastolic depolarization phase of the ionic pacemaker in lymphatic muscle cells (LMCs). Oscillating Ca2+ release from sarcoendoplasmic reticulum Ca2+ channels has been hypothesized to drive ANO1 activity during diastole, but the source of Ca2+ for ANO1 activation in smooth muscle remains unclear. Here, we investigated the role of the inositol triphosphate receptor 1 (Itpr1; Ip3r1) in this process using pressure myography, Ca2+ imaging, and membrane potential recordings in LMCs of ex vivo pressurized inguinal-axillary lymphatic vessels from control or Myh11CreERT2;Ip3r1fl/fl (Ip3r1ismKO) mice. Ip3r1ismKO vessels had significant reductions in contraction frequency and tone but an increased contraction amplitude. Membrane potential recordings from LMCs of Ip3r1ismKO vessels revealed a depressed diastolic depolarization rate and an elongation of the plateau phase of the action potential (AP). Ca2+ imaging of LMCs using the genetically encoded Ca2+ sensor GCaMP6f demonstrated an elongation of the Ca2+ flash associated with an AP-driven contraction. Critically, diastolic subcellular Ca2+ transients were absent in LMCs of Ip3r1ismKO mice, demonstrating the necessity of IP3R1 activity in controlling ANO1-mediated diastolic depolarization. These findings indicate a critical role for IP3R1 in lymphatic vessel pressure-dependent chronotropy and contractile regulation.

A dual-clock-driven model of lymphatic muscle cell pacemaking to emulate knock-out of Ano1 or IP3R

“…At [IP 3 ] = 0, the control and IP 3 R-KO traces become one. This matches fairly closely the experimental results ( Zawieja et al, 2023 ), which show that contraction frequency in IP 3 R1-KO vessels was between 17% and 27% of IP 3 R1 fl/fl control counterparts across the pressure range that was studied. Specifically, at the distending pressure of 3 cmH 2 O, IP 3 R1-KO frequency was 26.9% of the control (4.45 versus 16.53/min).…”

“…Comparing the performance of the model with the experimental findings in the accompanying paper ( Zawieja et al, 2023 ), the model performs particularly well with respect to the AP frequencies observed in control, Ano1-KO, and IP3R-KO conditions. However, in other respects, the agreement is less good.…”

“…Here, genetic means were again used to obtain IP 3 R knock-out using Myh11CreER T2 ;IP3R1 fl/fl (IP3R1ismKO) mice and equivalent non-activated Cre controls. The companion paper to this one ( Zawieja et al, 2023 ) gives full details.…”

TRPV4-Expressing Tissue-Resident Macrophages Regulate the Function of Collecting Lymphatic Vessels via Thromboxane A2 Receptors in Lymphatic Muscle Cells

“…We then utilized newly developed Python-based tools to perform automated tracking of inner and outside diameter from a given region of interest along the lymphatic segment and subsequent automated in-depth quantitative analyses of lymphatic contractility. Consistent with our previous studies 46,47,50–57 , these analyses included the calculation of mean values of contraction amplitude, contraction frequency, ejection fraction (EF), fractional pump flow (FPF), end-diastolic diameter (EDD), end-systolic diameter (ESD), and width of contraction. Following stimulation with GSK101, the pumping capacity of all lymphatic vessels from both male and female mice was significantly impaired, this was evident by the significantly decreased contraction amplitude, EF, and FPF.…”

Role of Sensory Nerves in Pulmonary Fibrosis

“…Deficits in lymphatic contractile activity contributing to lymphedema in the leg include irregular contractile rhythms and decreased contractile magnitude [ 189 ]. While little is known about lymphatic contractile activity in the lung, in other tissue beds, increases in intracellular Ca 2+ are important in driving lymphatic pacemaker activity and contraction, and these processes are reliant on inositol triphosphate (IP3) receptors and the Ca 2+ -activated Cl − channel TMEM16A [ 190 , 191 ]. TMEM16A is overexpressed in pulmonary arterial smooth muscle cells in patients with idiopathic PH and contributes to vasoconstrictor and vascular remodeling responses [ 192 ], with similar effects observed in rodent models of PH [ 193 , 194 ].…”