Lymphatic muscle cells are the innate pacemaker cells regulating mouse lymphatic collecting vessel contractions
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eLife assessment
This manuscript aims to identify the pacemaker cells in the lymphatic collecting vessels - the cells that initiate the autonomous action potentials and contractions needed to drive lymphatic pumping. Through the exemplary use of existing approaches (genetic deletions and cytosolic calcium detection in multiple cell types), the authors convincingly determine that lymphatic muscle cells are the origin of the action potential that triggers lymphatic contraction. This fundamental discovery establishes a new standard for the field of lymphatic physiology.
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Abstract
Collecting lymphatic vessels (cLVs) exhibit spontaneous contractions with a pressure-dependent frequency, but the identity of the lymphatic pacemaker cell is still debated. By analogy to pacemakers in the GI and lower urinary tracts, proposed cLV pacemaker cells include interstitial cells of Cajal like cells (ICLC), pericytes, as well as the lymphatic muscle (LMCs) cells themselves. Here we tested the extent to which these cell types are invested into the mouse cLV wall and if any cell type exhibited morphological and functional processes characteristic of pacemaker cells: a contiguous network; spontaneous Ca 2+ transients; and depolarization-induced propagated contractions. We employed inducible Cre (iCre) mouse models routinely used to target these specific cell populations including: c-kitCreER T2 to target ICLC; PdgfrβCreER T2 to target pericytes; PdgfrαCreER TM to target CD34 + adventitial fibroblast-like cells or ICLC; and Myh11CreER T2 to target LMCs. These specific inducible Cre lines were crossed to the fluorescent reporter ROSA26mT/mG, the genetically encoded Ca 2+ sensor GCaMP6f, and the light-activated cation channel rhodopsin2 (ChR2). c-KitCreER T2 labeled both a sparse population of LECs and round adventitial cells that responded to the mast cell activator compound 48-80. PdgfrβCreER T2 drove recombination in both adventitial cells and LMCs, limiting its power to discriminate a pericyte specific population. PdgfrαCreER TM labeled a large population of interconnected, oak leaf-shaped cells primarily along the adventitial surface of the vessel. Titrated induction of the smooth muscle-specific Myh11CreER T2 revealed a LMC population with heterogeneous morphology. Only LMCs consistently, but heterogeneously, displayed spontaneous Ca 2+ events during the diastolic period of the contraction cycle, and whose frequency was modulated in a pressure-dependent manner. Optogenetic depolarization through the expression of ChR2 by Myh11CreER T2 , but not PdgfrαCreER TM or c-KitCreER T2 , resulted in a propagated contraction. These findings support the conclusion that LMCs, or a subset of LMCs, are responsible for mouse cLV pacemaking.The presence and functionality of proposed pacemaker cells in collecting lymphatic vessels was tested with various mouse Cre models to drive expression of a recombination reporter ROSA26mT/mG, a genetically encoded Ca 2+ sensor GCaMP6f, or the optogenetic tool channel-rhodopsin2. Lymphatic CD34 + adventitial cells co-express PDGFRΑ + while cKit + cells are mast cells; and neither cell type demonstrated pacemaking functionality. Myh11CreER T2 identified lymphatic muscle cells which exhibited pacemaker behaviors such as pressure-dependent calcium events during diastole and propagated contraction induced by optical stimulation of channel-rhodopsin2.
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eLife assessment
This manuscript aims to identify the pacemaker cells in the lymphatic collecting vessels - the cells that initiate the autonomous action potentials and contractions needed to drive lymphatic pumping. Through the exemplary use of existing approaches (genetic deletions and cytosolic calcium detection in multiple cell types), the authors convincingly determine that lymphatic muscle cells are the origin of the action potential that triggers lymphatic contraction. This fundamental discovery establishes a new standard for the field of lymphatic physiology.
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Reviewer #1 (Public Review):
Summary:
This manuscript explores the multiple cell types present in the wall of murine-collecting lymphatic vessels with the goal of identifying cells that initiate the autonomous action potentials and contractions needed to drive lymphatic pumping. Through the use of genetic models to delete individual genes or detect cytosolic calcium in specific cell types, the authors convincingly determine that lymphatic muscle cells are the origin of the action potential that triggers lymphatic contraction.
Strengths:
The experiments are rigorously performed, the data justify the conclusions, and the limitations of the study are appropriately discussed.
There is a need to identify therapeutic targets to improve lymphatic contraction and this work helps identify lymphatic muscle cells as potential cellular targets for …
Reviewer #1 (Public Review):
Summary:
This manuscript explores the multiple cell types present in the wall of murine-collecting lymphatic vessels with the goal of identifying cells that initiate the autonomous action potentials and contractions needed to drive lymphatic pumping. Through the use of genetic models to delete individual genes or detect cytosolic calcium in specific cell types, the authors convincingly determine that lymphatic muscle cells are the origin of the action potential that triggers lymphatic contraction.
Strengths:
The experiments are rigorously performed, the data justify the conclusions, and the limitations of the study are appropriately discussed.
There is a need to identify therapeutic targets to improve lymphatic contraction and this work helps identify lymphatic muscle cells as potential cellular targets for intervention.
Weaknesses:
My only major comment would be that the manuscript provides a lot of rich information describing the cellular components of the muscular lymphatic vessel wall and that these data are not well represented by the title. The title (while currently accurate) could be tweaked to better represent all that is in this manuscript. Maybe something like "Characterization/Interrogation of the cellular components of murine collecting lymphatic vessels reveals that lymphatic muscle cells are the innate pacemaker cells regulating lymphatic contractions" or "Discovery/Confirmation of lymphatic muscle cells as innate pacemaker cells of lymphatic contraction through characterization of the cellular components of murine collecting lymphatic vessels". Potentially a cartoon summary figure of the components that make up the collecting lymphatic vessel wall could also be included. In my opinion, these changes will make this manuscript of more interest to a broader group of scientists. I have a few additional comments for consideration to improve the clarity and enhance the discussion of this work.
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Reviewer #2 (Public Review):
Summary:
This is a well-written manuscript describing studies directed at identifying the cell type responsible for pacemaking in murine-collecting lymphatics. Using state-of-the-art approaches, the authors identified a number of different cell types in the wall of these lymphatics and then using targeted expression of Channel Rhodopsin and GCaMP, the authors convincingly demonstrate that only activation of lymphatic muscle cells produces coordinated lymphatic contraction and that only lymphatic muscle cells display pressure-dependent Ca2+ transients as would be expected of a pacemaker in these lymphatics.
Strengths:
The use of a targeted expression of channel rhodopsin and GCaMP to test the hypothesis that lymphatic muscle cells serve as the pacemakers in musing lymphatic collecting vessels.
Weaknesses:
The …
Reviewer #2 (Public Review):
Summary:
This is a well-written manuscript describing studies directed at identifying the cell type responsible for pacemaking in murine-collecting lymphatics. Using state-of-the-art approaches, the authors identified a number of different cell types in the wall of these lymphatics and then using targeted expression of Channel Rhodopsin and GCaMP, the authors convincingly demonstrate that only activation of lymphatic muscle cells produces coordinated lymphatic contraction and that only lymphatic muscle cells display pressure-dependent Ca2+ transients as would be expected of a pacemaker in these lymphatics.
Strengths:
The use of a targeted expression of channel rhodopsin and GCaMP to test the hypothesis that lymphatic muscle cells serve as the pacemakers in musing lymphatic collecting vessels.
Weaknesses:
The only significant weakness was the lack of quantitative analysis of most of the imaging data shown in Figures 1-11. In particular, the colonization analysis should be extended to show cells not expected to demonstrate colocalization as a negative control for the colocalization analysis that the authors present.
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Reviewer #3 (Public Review):
Summary:
Zawieja et al. aimed to identify the pacemaker cells in the lymphatic collecting vessels. Authors have used various Cre-based expression systems and optogentic tools to identify these cells. Their findings suggest these cells are lymphatic muscle cells that drive the pacemaker activity in the lymphatic collecting vessels.
Strengths:
The authors have used multiple approaches to test their hypothesis. Some findings are presented as qualitative images, while some quantitative measurements are provided.
Weaknesses:
- More quantitative measurements.
- Possible mechanisms associated with the pacemaker activity.
- Membrane potential measurements. -
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