Mechanisms of Connexin-Related Lymphedema

Abstract

Rationale: Mutations in GJC2 and GJA1, encoding connexins (Cxs) 47 and 43 respectively, are linked to lymphedema, but the underlying mechanisms are unknown. Because efficient lymph transport relies on the coordinated contractions of lymphatic muscle cells (LMCs) and the electrical coupling of those through Cxs, Cx-related lymphedema is proposed to result from dyssynchronous contractions of lymphatic vessels. Objective: Determine which Cx isoforms in LMCs and/or lymphatic endothelial cells (LECs) are required for the entrainment of lymphatic contraction waves and efficient lymph transport. Methods and Results: We developed novel methods to quantify the spatiotemporal entrainment of lymphatic contraction waves and used optogenetic techniques to analyze calcium signaling within and between the LMC and LEC layers. Genetic deletion of the major LEC Cxs (Cx43, Cx47, or Cx37) revealed that none were necessary for the synchronization of the global calcium events that triggered propagating contraction waves. We identified Cx45 in human and mouse LMCs as the critical Cx mediating the conduction of pacemaking signals and entrained contractions. Smooth muscle-specific Cx45 deficiency resulted in 10–18-fold reduction in conduction speed, partial-to-severe loss of contractile coordination, and impaired lymph pump function ex vivo and in vivo. Cx45-deficiency resulted in profound inhibition of lymph transport in vivo, but only under an imposed gravitational load. Conclusions: Our results: 1) identify Cx45 as the Cx isoform mediating the entrainment of the contraction waves in LMCs; 2) show that major endothelial Cxs are dispensable for the entrainment of contractions; 3) reveal a lack of coupling between LECs and LMCs, in contrast to arterioles; 4) point to lymphatic valve defects, rather than contraction dyssynchrony, as the mechanism underlying GJC2- or GJA1-related lymphedema; and 5) show that a gravitational load exacerbates lymphatic contractile defects in the intact mouse hindlimb, which is likely critical for the development of lymphedema in the adult mouse.

Effects of Elevated Downstream Pressure and the Role of Smooth Muscle Cell Coupling through Connexin45 on Lymphatic Pacemaking

“…The recombination efficiency of this Smmhc-CreER T2 in lymphatic cells was previously assessed by crossing it with the fluorescent reporter ROSA26 mT/mG ; in that case, we showed recombination in >95% of the LMCs [ 18 ]. We also assessed the recombination efficiency specifically in Smmhc-CreER T2 ;Cx45 fx/fx , showing that Cx45 was successfully deleted from ~98% of the LMCs [ 7 ]. Cx45 fx/fx (no Cre) mice were also fed tamoxifen-containing chow for one week and used as one of the control groups; a second control group of WT mice were fed regular mouse chow.…”

“…The exposed tissue underneath the window was constantly perfused with warm (37 °C) Krebs solution. Once the preparation was stable, fluorescence videos were acquired and the spontaneous contractions of a collecting lymphatic were acquired at 10–20 fps through a 1x Zeiss macroscope objective using a Hamamatsu C11440 ORCA-Flash4.0 digital camera (2048 × 2048 pixels) [ 7 ]. Custom-written Python-based programs were used to automatically detect changes in diameter over time from a representative window/region of interest.…”

“…In healthy collecting lymphatics, the contractions of all LMCs within a lymphangion (the segment between consecutive valves) [ 6 ], and even across multiple lymphangions, are highly coordinated, resulting in a contraction wave that conducts at 8–10 mm/s [ 7 , 8 , 9 , 10 ]. The entrainment of LMC contraction waves occurs as a result of the rapid propagation of a pacemaking signal (i.e., action potential) from LMC to LMC through connexin (Cx) gap junctions along the lymphatic wall.…”

“…We recently demonstrated that strong electrical coupling between LMCs, with minimal interaction of other surrounding cell networks (e.g., limited electrical coupling between LMCs and lymphatic endothelial cells (LECs)), is essential for the focal generation of pacemaking signals and their rapid and efficient propagation along the lymphatic wall. Furthermore, the entrainment of lymphatic contractions is mediated primarily by LMC-LMC electrical communication through Cx45 gap junctions [ 7 ].…”

Simplified method to quantify valve back‐leak uncovers severe mesenteric lymphatic valve dysfunction in mice deficient in connexins 43 and 37

“…All video processing and two-dimensional analyses were performed using a set of custom-written Python-based programs. These analysis tools were developed from previously published methods for GI studies (Hennig et al 2010(Hennig et al , 2015Hennig, 2016) and recently adapted for lymphatic vessel studies (Castorena-Gonzalez et al 2018b).…”