Figures and data
Quantification of the F-P relationship.
Steps for quantitative assessment of pressure-induced chronotropy. A) Representative recording of pressure and diameter changes in a WT popliteal lymphatic vessel during inflow (Pin) and outflow (Pout) pressure steps between 0.5 and 5 cmH2O. Frequencies stated at the bottom of the trace were determined off-line using a custom peak detection program written in LabVIEW. B) Plot of frequency vs pressure for the vessel in A, showing how frequency reaches a plateau above 5 cmH2O. C) Fit of the frequency data with a first-order equation and calculation of the frequency difference (ΔF) between 5 and 0.5 cmH2O. D) Summary of contraction data for this representative WT vessel over the stated pressure range; see Methods for description of parameter calculations.
Consequences of ANO1 deletion and/or inhibition.
A) UMAP plot of Ano1 expression in the various cell populations within the walls of murine IALVs that were thoroughly cleaned prior to dissociation and scRNAseq analysis, as described in (Zawieja et al., 2025). The LMC cluster, composed of at least two subclusters of 978 cells, was identified by the expression of canonical smooth muscle cell markers Acta2 (SM α-actin), Myh11 (myosin heavy chain 11) and Itga8 (integrin alpha 8). Gray color in this and subsequent figures represents Acta2 expression, red/brown colors in this panel indicate Ano1 expression. Bubble plot shows Ano1 expression in the LMC and other cell clusters in terms of percent cells in an individual cluster expressing Ano1 (size of dot) and the Ano1 expression level relative to Acta2 (color of dot). Abbreviated names of the clusters are listed on the right. B) Slope of frequency vs pressure (F-P) for WT (C57Bl/6) vessels with / without treatment by the ANO1 inhibitor Ani9 (1 μM), for Ano1f/f control vessels, for Ano1 smKO vessels and for the latter treated with Ani9. C) Summary plots of contraction parameters as a function of pressure for the 5 respective groups of vessels. Horizontal bars indicate ranges of pressures with same specified statistical significance. WT N=15, n=20; WT+Ani9 N=5, n=6; Ano1 smKO N=14, n=23; Ano1f/f N=8, n=16; Ano1 smKO+Ani9 N=4, n=4.
Contractions of Ano1f/f and Ano1 smKO vessels.
A) Spontaneous contractions of an Ano1f/f (control) popliteal lymphatic vessel at various pressures. Average frequencies at each pressure are listed below the diameter traces. Frequency ranged from 2.1 min-1 at 0.5 cmH2O to 15.3 min-1 at 5 cmH2O. B) Spontaneous contractions of a Myh11-CreERT1;Ano1f/f (Ano1 smKO) popliteal vessel at various pressures. In this particular example, two additional pressure steps between 0.5 and 3 cmH2O were imposed (this example represents an early experiment before the protocol was finalized). Frequency ranged from 0.6 min-1 at 0.5 cmH2O to 2.2 min-1 at 5 cmH2O.
Consequences of Piezo1 deletion.
A) UMAP and bubble plots of Piezo1 and Piezo2 expression in the various IALV cell clusters. B) Slope of F-P relationship for popliteal lymphatics from Piezo1f/f, Piezo1 smKO, Piezo1 nestin KO, Piezo1 lecKO mice. C) Summary plots of contraction parameters as a function of pressure for the 4 respective groups of vessels. Piezo1f/f N=9, n=17; Piezo1 smKO N=7, n=13; Piezo1 nestin KO N=5, n=10; Piezo1 lecKO N=5, n=6.
Consequences of Trpc6 and/or Trpc3 deletion.
A) UMAP and bubble plots of Trpc6 and Trpc3 expression in the various IALV cell clusters. B) Slope of F-P relationship for popliteal lymphatics from SV129 controls, Trpc6-/-, Trpc3-/- and Trpc6-/-;Trpc3-/- mice. C) Summary plots of contraction parameters as a function of pressure for the 4 respective groups of vessels. SV129 control N=8, n=11; Trpc6-/- N=5, n=10; Trpc3-/- N=7, n=11; Trpc6-/-;Trpc3-/- N=5, n=10.
Consequences of Trpm4 deletion.
A) UMAP and bubble plots of Trpm4 expression in the various IALV cell clusters. B) Slope of F-P relationship for popliteal lymphatics from Trpm4f/f and Trpm4 smKO mice. C) Summary plots of contraction parameters as a function of pressure for the two respective groups of vessels. Trpm4 smKO N=6, n=13; Trpm4f/f N=4, n=7.
Consequences of Pkd2 (TRPP1) deletion.
UMAP and bubble plots of Pkd2 expression in the various IALV cell clusters. B) Slope of F-P relationship for popliteal lymphatics from Pkd2 f/f and Pkd2 smKO mice. C) Summary plots of contraction parameters as a function of pressure for the two respective groups of vessels. Pkd2 smKO N=6, n=13; Pkd2 f/f N=4, n=8.
Consequences of Trpv4 deletion.
UMAP and bubble plots of Trpv4 expression in the various IALV cell clusters. B) Slope of F-P relationship for popliteal lymphatics from Trpv4f/f and Trpv4 smKO mice. C) Summary plots of contraction parameters as a function of pressure for the two respective groups of vessels. TRPV4 smKO N=5, n=10; TRPV4f/f N=3, n=7.
Consequences of Trpv2 inhibition.
UMAP and bubble plots of Trpv2 expression in the various IALV cell clusters. B) Slope of F-P relationship for popliteal lymphatics from WT mice with/without treatment with the TRPV2 inhibitors tranilast (10 μM) and SET2 (1 μM). C) Summary plots of contraction parameters as a function of pressure for the three respective groups of vessels. WT+tranilast N=5, n=10; WT+SET2 N=3, n=6.
Consequences of ENaC inhibition.
UMAP and bubble plots of ENaC subunit expression in the various IALV cell clusters. B) Slope of F-P relationship for popliteal lymphatics from WT mice with/without treatment with the ENaC inhibitors amiloride (5 μM) and benzamil (1 μM). WT N=15, n=20, WT+amiloride N=5, n=9; WT+benzamil N=6, n=11.
Consequences of Slc8a1 (NCX1) deletion.
UMAP and bubble plots of Slc8a1 expression in the various IALV cell clusters. B) Slope of F-P relationship for popliteal lymphatics from Slc8a1f/f and Slc8a1 smKO mice. C) Summary plots of contraction parameters as a function of pressure for the two respective groups of vessels. Slc8a1f/f N=7, n=12; Slc8a1 smKO N=7, n=13.
Consequences of HCN inhibition.
UMAP and bubble plots of Hcn gene family expression in the various IALV cell clusters. B) Slope of F-P relationship for popliteal lymphatics from WT mice with/without treatment with the HCN inhibitors ivabradine (3 μM) and zatebradine (3 μM). WT N=15, n=20, WT+zatebradine. N=5, n=10; WT+ivabradine N=5, n=10.
Consequences of Kcnq1 deletion.
UMAP and bubble plots of Kcnq4 expression in the various IALV cell clusters. B) Slope of F-P relationship for popliteal lymphatics from WT and Kcnq4 smKO mice. C) Summary plots of contraction parameters as a function of pressure for the two respective groups of vessels. Kcnq4 smKO N=5, n=7.
Consequences of Kir2.x inhibition.
UMAP and bubble plots of Kcnj2 (Kir2.1), Kcnj12 (Kir2.2) and Kcnj8 (Kir6.1) expression in various IALV cell clusters. B) Slope of F-P relationship for popliteal lymphatics from WT mice with/without treatment with the Kir2 inhibitor BaCl2 (100 μM). WT N=15, n=20, WT+BaCl2 N=4, n=11.
Consequences of Cacna1c (Cav1.2) and Cacna1g/Cacna1h (Cav3.1/3.2) deletion.
UMAP and bubble plots of the expression of voltage-gated Ca2+ channels and their accessory subunits in the various IALV cell clusters. B) Slope of F-P relationship for popliteal lymphatics from Cacan1cf/f, Cacna1c smKO and Cacna1g-/-;Cacna1h-/- mice. C) Summary plots of contraction parameters as a function of pressure for the three respective groups of vessels. Horizontal bar indicates range of pressures with same specified statistical significance. Cacna1c: alpha-1c, Cacna2d1: α2/δ1, Cacnb2: β2, Cacnb3: β3, Cacna1d: alpha-1d (Cav1.3), Cacna1a: Cav2.1 (P/Q), Cacna1g: Cav3.1, Cacna1h: Cav3.2. Cacna1cf/f N=10, n=20; Cacna1c smKO N=14, n=24; Cacna1g-/-;Cacna1h-/-N=16, n=25.
Consequences of Itpr1 (IP3R1) deletion.
UMAP and bubble plots of Ip3r1 expression in the various IALV cell clusters. B) Slope of F-P relationship for popliteal lymphatics from Itpr1f/f and Itpr1 smKO mice. C) Summary plots of contraction parameters as a function of pressure for the two respective groups of vessels. Horizontal bar indicates range of pressures with same specified statistical significance. Itpr1 smKO N=5, n=8; Itpr1f/f N=5, n=10.
Contractions of IP3R1f/f and IP3R1 smKO vessels.
A) Spontaneous contractions of an IP3R1f/f(control) popliteal lymphatic vessel at various pressures. Average frequencies at each pressure are listed below the diameter traces. Frequency ranged from 7.1 min-1 at 0.5 cmH2O to 17.9 min-1 at 5 cmH2O. B) Spontaneous contractions of an Myh11-CreERT2;IP3R1f/f (IP3R1 smKO) popliteal lymphatic vessel at various pressures. Frequency ranged from 0 min-1 at 0.5 cmH2O to 1.9 min-1 at 5 cmH2O.
Consequences of Gnaq and/or Gna11 deletion.
UMAP and bubble plots of Gnaq and Gna11 expression in the various IALV cell clusters. B) Slope of F-P relationship for popliteal lymphatics from Gnaqf/f;Gna11+/+, Myh11-CreERT2;Gnaqf/f (Gnaq smKO), Gna11-/- and Myh11-CreERT2;Gnaqf/f;Gna11-/- DKO mice. C) Summary plots of contraction parameters as a function of pressure for the four respective groups of vessels. Gnaqf/f;Gna11+/+ control N=3, n=6; Gnaq smKO N=4, n=8; Gna11-/- N=3, n=6; Myh11-CreERT2;Gnaqf/f;Gna11-/-N=8, n=13.
Examples of contractions of Gnaqf/f;Gna11+/+ and Myh11-CreERT2;Gnaqf/f;Gna11-/- DKO vessels.
A) Spontaneous contractions of a Gnaqf/f;Gna11+/+ (control) popliteal lymphatic vessel at various pressures. Average frequencies at each pressure are listed below the diameter traces. Frequency ranged from 1.6 min-1 at 0.5 cmH2O to 16.2 min-1 at 5 cmH2O. B) Spontaneous contractions in a Myh11-CreERT2;Gnaqf/f;Gna11-/- DKO popliteal vessel at various pressures. Frequency ranged from 0 min-1 at 0.5 cmH2O to 1.3 min-1 at 5 cmH2O. The noisy diameter signal at P=8, 10 cmH2O is caused by tracking glitches and does not represent high frequency contractions.
Consequences of Gna12/Gna13 deletion.
UMAP and bubble plots of Gna12 and Gna13 expression in the various IALV cell clusters. B) Slope of F-P relationship for popliteal lymphatics from Gna12f/f;Gna13+/+ and Myh11-CreERT2;Gna12f/f;Gna13-/- DKO mice. C) Summary plots of contraction parameters as a function of pressure for the two respective groups of vessels. Gna12f/f;Gna13+/+ controls N=5, n=10; Myh11-CreERT2;Gna12f/f;Gna13-/-N=7, N=15.
Bubble plot representing the results of scRNAseq analysis of the most highly expressed GPCRs in the various cell types.
Rows 1-10 show the canonical markers for LMCs (rows 1-4), LECs (rows 5-7) and AdvCs (rows 8-10), respectively. Rows 11-30 show the top 20 GPCRs expressed in LMCs, ranging from 87.5% of LMCs expressing Npy1R to 36.6% LMCs expressing F2R. Putative mechanosensitive GPCRs in other cell types (e.g. arterial SM), but with very low or absent expression in LMCs, are shown in rows 34-41; AT1R subtypes are shown in rows 32-33, cysteinyl leukotriene receptor subtypes in rows 34-35 and P2Y receptor subtypes in rows 36-37. Four other GPCRs are shown in rows 38-41. G-coupling proteins are shown in rows 42-47 and β-arrestins in rows 48-49. Red arrows at left are the GPCRs that were tested with inhibitors in the present study or in previous studies (see text for references).
Screening the effects of selective GPCR inhibitors.
Pacemaking frequency is compared between control and inhibitor (or knock out) at a single pressure, 3 cmH2O. Gray symbols: comparisons between Ano1f/fcontrols and Ano1 smKO, WT ± Ani9, IP3R1f/f controls and IP3R1 smKO, Gqf/f;G11+/+controls and Gq/11 DKO, Gq+/+;G11+/+ controls ± YM254890, showing that each treatment produced a very significant reduction in frequency using this test. Red symbols: comparisons between WT controls ± respective inhibitors for the various GPCR. None of the GPCR inhibitors produced a significant decrease in frequency, but the S1PR1 and AT1R inhibitors instead significantly increased frequency. Inhibitor concentrations were as follows: Gq/11 inhibitor YM254890 (100 nM); ANPR inhibitor AP811 (1 μM); ET1AR inhibitor BQ123 (1 μM); NPYR inhibitor BIBO3304 (1 μM); TXA2R inhibitor S18886 (20 nM); S1PR1 inhibitor SEW-2871 (1 μM); AT1R inhibitor losartan (10 μM). Comparisons between controls and KO vessels were made using 2-way ANOVAs with Dunnett’s post-hoc tests. Comparisons between controls ± inhibitors were made using paired t-tests. Ano1 smKO N=14, n=23; Ano1f/f N=8, n=16. WT N=15, n=20; WT ± Ani9 N=5, n=6. Itpr1 smKO N=5, n=8; Itpr1f/f N=5, n=10. Gnaqf/f;Gna11+/+control N=3, n=6; Myh11-CreERT2;Gnaqf/f;Gna11-/- N=8, n=13. Gq+/+;G11+/+ control ± YM254890 N=8, n=8. WT ± AP811 N=8, n=8. WT ± BQ123 N=7, n=7. WT ± BIBO3304 N=8, n=8. WT ± SEW-2871 N=4, n=4. WT ± losartan N=5, n=10. Agtr1a-/- N=3, n=6. S18886 N=4; n=11.
Genes for which deletion/inhibition produced significant attenuation or enhancement of the F-P relationship.
Consequences of deletion/inhibition of ion channels on contraction amplitude and/or tone.*
Proposed mechanotransduction mechanism underlying pressure-induced lymphatic chronotropy.
Summary diagram depicting the primary signaling pathway through which changes in pressure regulate the frequency of the ionic pacemaker in LMCs. Gq/11-coupled GPCRs are the primary, but perhaps not the exclusive mechanosensor; a role for G14-coupled GPCRs is possible. Multiple GPCRs, which remain to be identified, may transduce different pressure ranges. ANO1 is the primary, but perhaps not the exclusive effector that regulates activation of Cav1.2 channels, but the pacemaker will cycle at a basal rate in the absence of ANO1 input. Kv11 provides at least one source of repolarizing signal (Kim et al., 2023).
Comparisons of frequency at different pressure levels in IALVs for the various Gq/11 genotypes.
Data were analyzed using a mixed model ANOVA with Fischer’s LSD post hoc tests. * = Gq+/+;G11+/+ control vs Gq11-/-; * = Gq+/+;G11+/+ control vs Gq/11 DKO; * = G11-/-vs Gq/11 DKO. Gq+/+;G11+/+ control N=9, n=9; Gq11-/- N=13 n=13; Gq/11 DKO N=14 n=19.
