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

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 Myh11CreER^T2;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.