“Experimental and modeling insights into the factors determining the initiation and entrainment of lymphatic contractions”
Abstract
Contractions of a lymphangion, the segment between two one-way lymphatic valves, generate the pressure gradients that propel lymph back to the circulation. Each lymphangion is comprised of an inner sheet of lymphatic endothelial cells circumscribed by one or more layers of lymphatic muscle cells (LMCs). Each contraction is produced by an LMC action potential (AP) that propagates via gap junctions along the lymphangion. Yet, electrical coupling within and between cell layers and its impact on contraction waves and AP waves is poorly understood.
We combine studies in rat and mouse lymphatic vessels with mathematical modeling to show that initiation of AP waves depends on high input resistance (low current drain), whereas propagation depends on morphology and sufficient LMC:LMC coupling through connexins. The use of mice deficient in specific connexin isoforms allows us to determine that the major lymphatic endothelial cell (LEC) connexins, Cx37, Cx43, Cx47 are dispensable for entrained contraction and AP waves. This finding is surprising given that patients with Cx43 or Cx47 mutations develop lymphedema. Measurements of LMC and LEC membrane potential and Ca2+ imaging in specific cell layers suggest that the LMC and LEC layers are electrically uncoupled, which contrasts with tight coupling in the arterial wall.
Simulations show that 1) myoendothelial coupling must be low to facilitate AP generation and sustain an experimentally measured cross-junctional potential difference of 25 mV, allowing AP waves to propagate only along the LMC layer; 2) LMC:LMC resistance is estimated around 2–10 MW but depends on vessel structure and cell-cell coupling, such that some degree of LMC overlap protects AP waves against LMC decoupling; 3) the propensity of AP wave initiation is highest around the valves, where the density of LMCs is lower; and 4) a single pacemaker cell embedded in the LMC layer must be able to generate very large currents to overcome the current drain from the layer. However, the required current generation to initiate an AP wave is reduced upon stimulation of multiple adjacent LMCs. With stimulation of all LMCs, AP waves can also arise from heterogeneity in the electrical activity of LMCs. These findings advance our understanding of the electrical constraints that underlie initiation of APs in the LMC layer and make testable predictions about how morphology, LMC excitability, and LMC:LMC electrical coupling interact to determine the ability to initiate and propagate AP waves in lymphatic vessels.
Research Description
The experimental approaches used in Davis’ laboratory — isolated, perfused microvessel methods and single-cell electrophysiology — are combined with a variety of imaging techniques, including confocal microscopy, atomic force microscopy and total-internal reflectance fluorescence microscopy.Molecular analyses, such as site-directed mutagenesis and co-immunoprecipitation, is also used to identify the specific integrin-associated proteins that are involved in the modulation of ion channels and to identify the specific domains on the channels that are required for their regulation.