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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., (© 2023 Zawieja et al.)

We identified a regional dichotomy in murine lymphatic contractile function with regard to vessel location within the periphery or visceral cavity. All vessels isolated from peripheral regions [cervical, popliteal, inguinal, axillary, and internodal inguinal axillary (Ing-Ax)] developed robust contractions with maximal ejection fractions (EFs) of 50-80% in our ex vivo isobaric myograph experiments. Conversely, vessels isolated from the visceral cavity (mesenteric, thoracic duct, and iliac) demonstrated maximal EFs of ≤10%. Using pressure myography, sharp electrode membrane potential recordings, and Ca 2+ imaging, we assessed the role of L-type Ca 2+ channels in this contractile dichotomy. Ing-Ax membrane potential revealed a ~2-s action potential (AP) cycle (resting -35 mV, spike -5 mV, and plateau -11 mV) with a plateau phase that was significantly lengthened by the L-type Ca 2+ channel agonist Bay K8644 (BayK; 100 nM). APs recorded from mesenteric vessels, however, displayed a slower upstroke and an elongated time over threshold. BayK (100 nM) increased the mesenteric AP upstroke velocity and plateau duration but also significantly hyperpolarized the vessel. Contractions of vessels from both regions were preceded by Ca 2+ flashes, detected with a smooth muscle-specific endogenous Ca 2+ reporter, that typically were coordinated over the length of the vessel. Similar to the membrane potential recordings, Ca 2+ flashes in mesenteric vessels were weaker and had a slower rise time but were longer lasting than those in Ing-Ax vessels. BayK (100 nM) significantly increased the Ca 2+ transient amplitude and duration in both vessels and decreased time to peak Ca 2+ in mesenteric vessels. However, a higher concentration (1 μM) of BayK was required to produce even a modest increase in EF in visceral lymphatics, which remained at <20%. NEW & NOTEWORTHY Lymphatic collecting vessels isolated from murine peripheral tissues, but not from the visceral cavities, display robust contractile behavior similar to lymphatic vessels from other animal models and humans. These differences are partially explained by L-type Ca 2+ channel activity as revealed by the first measurements of murine lymphatic action potentials and contraction-associated Ca 2+ transients.