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

Abstract

Lymphatic vessels rely on spontaneous lymphatic muscle cell (LMC) contractions and one-way intraluminal valves to efficiently pump lymph and return it into the bloodstream. Intraluminal pressure is known to regulate the contractile function of lymphatics, with pressure elevation leading to increased contraction frequency and decreased amplitude. Contractions are normally initiated by a dominant pacemaker and are highly entrained among strongly coupled LMCs. Previously, we found that connexin45 is the major connexin isoform mediating LMC-LMC electrical coupling. Lymphatics from mice lacking smooth muscle connexin45 display uncoordinated, impaired contractions. Here, we utilized this connexin45-deficient model, pressure myography, and recently developed, novel analytical tools to assess the effects of elevated downstream pressure on the number, location, and frequency of lymphatic pacemakers. Our results show that, in vessels from healthy controls, an increase in downstream pressure resulted in the recruitment/development of new pacemakers and increased contractile frequency while a dominant pacemaker continued to be observed. In contrast, vessels from connexin45-deficient mice displayed significantly more pacemakers, but none were dominant; this worsened with elevated downstream pressure. These results suggest a potential protective mechanism through which the lymphatic vasculature adapts to transient increases in downstream pressure, but which may not be sustained in scenarios with chronic elevated downstream pressure.

A 1D model characterizing the role of spatiotemporal contraction distributions on lymph transport

“…Lastly, it is important to mention that this study did not specifically examine the probability of contraction patterns in a lymphatic vessel. Investigating the likelihood of dominant pacemaking locations would require a separate set of simulations 10 . As of now, there is no experimental data available on the contribution of each pacemaking location to the resulting contraction magnitude.…”

“…These specific sites are referred to as “pacemaking sites” or “pacemakers”. In addition to identifying these cells, various metrics are typically measured to study the contractions, such as contraction frequency and propagation speed and pacemaking distance 9 , 10 . These studies reveal that contractions exhibit wave-like behavior, highlighting the necessity of employing a 1-D modeling approach to investigate the lymphatic system.…”

“…It is increasingly clear that the timing of lymphatic contractions may be governed by pacemaking cells within the lymphatics. Pacemaking cells serve as the sites of initiation for the spontaneous transient depolarization that drives lymphatic contraction and electrically propagates to adjacent lymphatic muscle cells 9 , 10 .…”

A 1D Model Characterizing the Role of Spatiotemporal Contraction Distributions on Lymph Transport

“…These locations are called pacemakers or pacemaking sites. Besides locating these cells, some metrics are usually measured to study the contractions such as contraction propagation speed, pacemaking distance, etc 34,35 . From these studies, we realize that contractions demonstrate a wave behavior which explains why a one-dimensional model is required to study the lymphatic system.…”

Connexins, Innexins, and Pannexins: From Biology to Clinical Targets

“…Cx45 is also expressed in the heart, but additionally seems to play a prominent role in the lymphatic system, for example, by mediating lymphatic muscle cell coupling. Castorena-Gonzalez et al [ 21 ] used their Cx45-deficient mouse model to show how this connexin isoform regulates lymphatic pacemaking upon elevated downstream pressure. The more recently described pannexin channel proteins also seem to play a role in the vasculature, with multiple studies in mice pointing to a pathological role for Panx1.…”