Myh11-CreER^T2 mice [B6.FVB-Tg (Myh11-cre/ERT2)1Soff/J] were originally obtained from Stefan Offermanns (Max-Planck Institute, Bad Neuheim). Itga8-CreER^T2 mice were obtained from Joe Miano and Lin Gan (Medical College of Georgia at Augusta University [Warthi et al., 2022)]. Both strains were bred with Ca _v 1.2 ^f/f mice (Cacna1c ^tm3Hfm/J; JAX #024714) or Rosa26mT/mG ^f/f reporter mice (JAX, #007676), to generate Myh11-CreER^T2 ;Ca _v 1.2 ^f/f , Itga8-CreER^T2 ;Ca _v 1.2 ^f/f , Myh11-CreER^T2 ;Rosa26mT/mG ^f/f , and Itga8-CreER^T2 ;Rosa26mT/mG ^f/f mice. The offspring were injected with tamoxifen (10 mg/ml, 100µl i.p.) for consecutive 5 days and allowed to recover for at least 10 days before testing, as previously described (To et al., 2020). Ca _v 1.2 ^f/f mice were induced using the same protocol. All genotypes were verified by PCR. Mice (18–30 g) of either sex were studied at 2–6 months of age.

Mice were anesthetized by intraperitoneal injection of Ketamine/Xylazine (100/10 mg/kg) and placed in the prone position on a heated tissue dissection/isolation pad. The superficial saphenous vein was exposed by a proximal-to-distal incision in the skin over the calf and the popliteal afferent lymphatic vessels on each side of the vein were identified and isolated as previously described (Castorena-Gonzalez et al., 2018b). Each vessel was then pinned with short segments of 40 µm stainless steel wire onto a Sylgard-coated dissection chamber filled with BSA-containing Krebs buffer at room temperature. Once secured, the surrounding adipose and connective tissues were removed by microdissection. An isolated vessel was then transferred to a 3-ml observation chamber on the stage of a Leica inverted microscope, cannulated and pressurized to 3 cm H₂O using two glass micropipettes (50–60 µm outside diameter). With the vessel pressurized, the segment was cleared of remaining connective and adipose tissue. Polyethylene tubing was attached to the back of each glass micropipette and connected to a computerized pressure controller (Scallan et al., 2013), with independent control of inflow pressure (Pin) and outflow pressure (Pout). To minimize diameter-tracking artifacts associated with longitudinal bowing at higher intraluminal pressures, input and output pressures were briefly set to 10 cm H₂O at the beginning of every experiment, and the vessel segment was stretched axially to remove any longitudinal slack.

Following this procedure, each lymphatic vessel was allowed to equilibrate at 37°C with inflow and outflow pressures set to 3 cm H₂O. Constant exchange of Krebs buffer was maintained using a peristaltic pump at a rate of 0.5 ml/min. After temperature stabilized, the vessel typically began to exhibit spontaneous contractions within 10–15 min that stabilized in a consistent pattern within ∼30 min. Custom LabVIEW programs (National Instruments; Austin, TX) acquired real-time analog data and digital video through an A-D interface (National Instruments) and detected the inner diameter of the vessel (Davis, 2005). Videos of the contractile activity of lymphatic vessels were recorded for further analyses under bright-field illumination at 30 fps using a firewire camera (Basler, Graftek Imaging; Austin, TX).

The contractile parameters of each vessel were characterized at different levels of intraluminal pressure spanning the physiological range from 0.5 to 10 cm H₂O (in successive steps: 3, 2, 1, 0.5, 3, 5, 8, and 10 cm H₂O). Spontaneous contractions were recorded at each pressure over intervals of 2–3 min. Wild type popliteal lymphatics typically exhibited their strongest contractions at pressures between 1-2 cm H₂O and contraction frequency increased 3 to 5-fold over the pressure range tested. During the pressure response protocol, both the input and output pressures were maintained at equal levels so that there was no imposed pressure gradient for forward flow. At the end of every experiment, all vessels were equilibrated by perfusion with calcium-free Krebs buffer containing 3 mM EGTA for 30 min, and passive diameters were obtained at each level of intraluminal pressure.

Once an experiment was completed, internal diameter traces (from a single representative region of interest) and/or videos of spontaneous contractions were analyzed using custom-written LabVIEW programs to detect end diastolic diameter (EDD), end systolic diameter (ESD), and contraction frequency (FREQ, computed on a contraction-by-contraction basis and averaged over a 2–3 min period). These data were used to calculate commonly reported parameters that characterize the lymphatic contractile function: A m p l i t u d e   A M P = E D D − E S D (1) N o r m a l i z e d   A m p l i t u d e = A M P / D MAX (2) E j e c t i o n   F r a c t i o n   E F = E D D 2 − E S D 2 E D D 2 (3) T o n e = D MAX − E D D D MAX × 100 (4) F r a c t i o n a l   P u m p   F l o w   F P F = E F ∙ F R E Q (5) where D_MAX represents the maximum passive diameter (obtained after 30-min equilibration in calcium-free Krebs solution) at a given level of intraluminal pressure. Each of these contractile parameters represents the average of all the recorded contractions during the 2–3 min time period corresponding to each level of intraluminal pressure. In control vessels, contraction amplitudes at pressures between 0.5 and 5 cm H₂O were typically between 20 and 60 μm for a maximal passive diameter of 60–100 μm. In Cav1.2-deficient vessels of comparable size, the changes in inner diameter typically were <5 µm and contraction waves (to the extent they were present) were not entrained, i.e., the diameter fluctuations resembled the irregular vasomotion often observed in lymphatic diastole or in arteries. To exclude these events, a value of 5 µm was used as the threshold for determining a valid spontaneous contraction (for determination of FREQ).

Active lymphatic contractions are critical for pumping lymph against adverse intraluminal pressure gradients (Scallan et al., 2016). To assess the impact of Cav1.2 deletion on the pump strength of single lymphangions, a second protocol was conducted in segments containing exactly 2 valves, similar to that described previously (Davis et al., 2012; Lapinski et al., 2017; Castorena-Gonzalez et al., 2018a). With Pin held at 1 cm H₂O, Pout was elevated ramp-wise from 1 to 12 cm H₂O at a rate of ∼3 cm H₂O/min while monitoring the behavior of the outflow valve. The outflow valve normally opened and closed during each contraction cycle, alternating with the inflow valve (Davis et al., 2011), but when Pout exceeded the level of internal pressure generated by a spontaneous contraction, the outflow valve failed to open during lymphatic systole. The Pout value at this point in time (minus Pin) corresponded to the “pump limit” of the lymphangion (Lapinski et al., 2017; Castorena-Gonzalez et al., 2018a). We previously showed that simultaneous measurements of valve position and internal pressure (Psn, made near the output valve using a servo-nulling micropipette), confirm that the pump limit, as assessed by the outflow valve behavior, corresponds to the amplitude of the internal pressure spike at the point in time when Psn fails to exceed the value of Pout. Thus, pump limit can be determined from the outflow valve behavior without directly measuring internal pressure.

A variation of this protocol was used in certain instances in which the outflow valve “locked” open during diastole, a behavior that occurs unpredictably, as described in a previous publication (Bertram et al., 2017). Outflow valve “lock” occurs in some vessels during an increasing Pout ramp when the inflow valve remains closed throughout diastole, dictating that the outflow valve remains open (“locked”) throughout the contraction cycle. This behavior is not directly related to pump strength but rather to the chance (50%) that the inflow valve has a lower ΔP (Pout—Pin) for closure than the outflow valve at any particular diastolic pressure (Davis et al., 2011). To circumvent this problem in the subset of vessels exhibiting the behavior, we devised an alternate protocol. When the outflow valve transiently closed during lymphatic diastole, due to the suction effect of vessel wall expansion (Jamalian et al., 2017), Pout was stepped rapidly from 1 to 12 cm H₂O, forcing the outflow valve to remain closed unless the internal pressure generated during systole was sufficient to overcome the level of Pout. Pout was then slowly lowered, ramp-wise, at ∼3 cm H₂O/min (or in some cases in step-wise increments of 0.5 cm H₂O) while spontaneous contractions continued to occur, typically at an elevated rate due to the higher Pout level (Scallan et al., 2012). The valve would then open during lymphatic systole at the point corresponding to the pump limit. We confirmed in test vessels that the pump limit determined by this procedure was very close (within 0.3 cm H₂O) to that determined by the increasing Pout ramp method.

Valve position, either open or closed, was determined from replay of the recorded protocol videos using a LabVIEW program, as described previously (Davis et al., 2012), while maintaining synchronization of the valve position data with the pressure and diameter data. The pump limit was then determined as described above. Pump tests on each vessel were performed at various levels of Pin (which determines the preload) in duplicate or triplicate and the values at each given level of Pin were averaged to give a single pump limit to be used for statistical analysis.

Total RNA was extracted from dissected and cleaned inguinal axillary lymphatic vessels using the Arcturus PicoPure RNA isolation kit with on-column DNase I treatment (Qiagen, Valencia, CA) according to the manufacturer’s instructions. RNA was eluted with 25 μl nuclease-free water. Purified RNA was transcribed into cDNA using High-Capacity cDNA Reverse Transcription kit (ThermoFisher Scientific, Waltham, MA). Real-time PCR was performed on cDNAs prepared from each sample using 2x PrimeTime Gene Expression Master Mix (IDT, Coralville, IA) with predesigned TaqMan probes: Mm.PT.58.33257376.gs for β-actin and Mm.PT.58.12188337 for Cacna1c (IDT, Coralville, IA). The Cacna1c primer targeted a sequence in the floxed region of Cav1.2. Real-time PCR protocols were as follows: preheating at 95°C for 3 min, 45 cycles of two-step cycling of denaturation at 95°C for 15 s and annealing/extension steps of 30 s at 60°C. Data collection was carried out using a Bio-Rad CFX 96 Real-Time Detection System (software version Bio-Rad CFX Manager 3.1; Bio-Rad, Hercules, CA, United States). The relative expression level was calculated according to the published 2^−ΔΔCT method (Livak and Schmittgen, 2001).

For assessment of smooth muscle recombination efficiency, cannulated and pressurized popliteal lymphatic vessels were equilibrated in calcium-free solution for 15 min to prevent movement during live imaging. Image stacks were acquired with a Yokagawa CSU-X Spinning Disc Confocal Microscope on an inverted Olympus IX81 with a Hamamatsu Flash4 camera and ×20 dry objective using Metamorph software. Each live vessel acquisition created a Z-stack of images in 1 micron increments from the lower surface of the vessel to its midpoint for both the membrane GFP (488 nm) and membrane tdTomato wavelengths (561 nm).

To assess recombination efficiency in the smooth muscle layer of popliteal lymphatic vessels, we assessed the percent area of the popliteal vessels covered by GFP⁺ lymphatic muscle cells. Maximum projections were made of the GFP and tdTomato image stacks using Image J. A mask of the GFP⁺ lymphatic muscle cells was created, despeckled, and then was used to assess the percent area of the tdTomato maximum projection encompassed by the mask using the Image J threshold and area measure function.

Krebs buffer contained: 146.9 mM NaCl, 4.7 mM KCl, 2 mM CaCl₂·2H₂O, 1.2 mM MgSO₄, 1.2 mM NaH₂PO₄·H₂O, 3 mM NaHCO₃, 1.5 mM Na-HEPES, and 5 mM D-glucose (pH = 7.4). An identical buffer was prepared with the addition of 0.5% bovine serum albumin (“Krebs-BSA”). During cannulation Krebs-BSA was present both luminally and abluminally; during the experiment the abluminal solution was constantly exchanged with Krebs buffer. Ca²⁺-free Krebs was Krebs with 3 mM EGTA replacing CaCl₂·2H₂O and used to determine passive diameter. All chemicals were obtained from Sigma-Aldrich (St. Louis, MO), with the exception of BSA (US Biochemicals; Cleveland, OH), MgSO₄ and Na-HEPES (ThermoFisher Scientific; Pittsburgh, PA).

Statistical analyses were performed using Prism9 (Graphpad Software, San Diego, CA). The number n refers to the number of vessels included per group. The normality of each data set was first tested to determine whether parametric or non-parametric tests should be used. Statistical differences in the various contraction parameters were tested at each pressure level between male and female vessels and between Cav1.2 ^f/f and Itga8-CreER^T2 ;Cav1.2 ^f/f vessels using two-way ANOVAs with repeated measures and Tukey’s multiple comparison tests. A one-way ANOVA with Kruskal-Wallis non-parametric tests and Dunn’s multiple comparison tests were used for the analysis of pump tests, as 3 of the 4 tests for normality in Prism indicated that the pump test data were not normally distributed. Data are plotted as mean ± SEM with significance set at p < 0.05 unless otherwise stated.

We compared the survival of Itga8-CreER^T2 ;Cav1.2^f/f mice with Myh11-CreER^T2 ;Cav1.2 ^f/f mice after the start of the tamoxifen injection protocol (Figure 1). Myh11-CreER^T2 ;Cav1.2 ^f/f mice began to die ∼day 15 after the first injection and no Myh11-CreER^T2 ;Cav1.2 ^f/f mice survived past day 20 post-induction, compared to 100% (5/5) male Itga8-CreER^T2 ;Cav1.2 ^f/f mice that were still alive at the time of sacrifice on day 78–82 post-induction and (1/1) female Itga8-CreER^T2 ;Cav1.2 ^f/f mice that was alive at the time of sacrifice on day 80 post-induction. Additionally, all Itga8-CreER^T2 ;Cav1.2 ^f/f mice were in good health and continued to feed and gain weight normally up until the day of sacrifice.

We recently reported the efficiency of recombination for the Rosa26 mT/mG reporter in lymphatics from Myh11-CreER^T2 ;Rosa26mT/mG ^f/f vs. Itga8-CreER^T2 ;Rosa26mT/mG ^f/f mice [Figure 3 in ref (Warthi et al., 2022)]. Here, we reanalyzed a subset of that data (2 male Itga8-CreER^T2 ;Rosa26mT/mG ^f/f mice) along with data from two (new) female Itga8-CreER^T2 ;Rosa26mT/mG ^f/f mice to compare the recombination efficiency in lymphatics from mice of different sexes. Fluorescence images of GFP in pressurized popliteal lymphatics are shown in Figure 2A. GFP expression is evident in the circumferential (i.e., smooth muscle) layer of cells in both male and female vessels. Quantification of recombination efficiency was similar between male and female vessels, as shown in Figure 2B. Because it is well known that recombination efficiency depends not only on the particular Cre used, but also on the length of the floxed gene (Stifter and Greter, 2020), we assessed the degree of Cav1.2 knock down by Itga8-CreER^T2 in male and female inguinal-axillary lymphatics using qPCR. As quantified in Figure 2C, Cav1.2 levels were decreased by ∼75% from control in both male and female lymphatic vessels. The actual degree of knock down of Cav1.2 may have been even greater because the qPCR results represent message from all cell types in the vessel wall that may also express Cav1.2, including non-SM cells such as neurons and some immune cells.

The absence of propulsive, spontaneous contractions in popliteal lymphatic vessels from Myh11-CreER^T2 ;Cav1.2 ^f/f mice was described in a previous publication (To et al., 2018). In that study, normal contractions, with amplitudes of 40–60 μm at pressures 0.5–3 cm H₂O, were recorded in 7 of 7 popliteal vessels from Cav1.2 ^f/f mice but were absent in 5 of 5 vessels from Myh11-CreER^T2 ;Cav1.2 ^f/f mice [see Figure 12 in (To et al., 2018)]. The residual contractile activity observed in Myh11-CreER^T2 ;Cav1.2 ^f/f vessels consisted only of small (<5 μm amplitude), non-entrained diameter oscillations with an irregular frequency, and neither the frequency nor amplitude were highly regulated by pressure as in WT or Cav1.2 ^f/f vessels (To et al., 2018). However, a subsequent analysis of popliteal lymphatic contractions in another cohort of Myh11-CreER^T2;Cav1.2^f/f mice, studied at the same time point after tamoxifen induction, revealed that 2 of 7 vessels did exhibit small (5–12 μm) contractions at certain low pressures (Zawieja et al., 2022).

We analyzed the contractile activity of popliteal lymphatic vessels from Itga8-CreER^T2 ;Cav1.2 ^f/f mice and a new cohort of Cav1.2 ^f/f mice after tamoxifen induction. Representative examples of spontaneous contractions observed in vessels from the two strains of mice are shown in Figure 3. Robust contractions were observed at all pressures between 0.5 and 10 cm H₂O in popliteal vessels from Cav1.2 ^f/f mice, similar to results we have reported in multiple studies from other tamoxifen-treated control vessels (Castorena-Gonzalez et al., 2018a; To et al., 2018; Zawieja et al., 2018). The contraction amplitude in the Cav1.2 ^f/f vessel shown in Figure 3A varied between 44 and 64 μm depending on the pressure level, with a maximum amplitude at 2 cm H₂O. As before, contraction frequency was highly sensitive to pressure such that frequency decreased from 12 min⁻¹ at 3 cm H₂O to 3 min⁻¹ at 0.5 cm H₂O. In this vessel, a maximal frequency of 14 min⁻¹ was reached at 5 cm H₂O; thus, frequency varied 4.6-fold over this pressure range. In contrast, the popliteal lymphatic from an Itga8-CreER^T2 ;Cav1.2 ^f/f mouse only exhibited contractions less than 3 μm in amplitude at all pressures (Figure 3B), with an estimated diameter oscillation frequency of ∼10 min⁻¹ at all pressures (see insert showing zoomed diameter trace).

The average contraction parameters from popliteal lymphatic vessels from 4 male and 5 female Cav1.2 ^f/f mice and from 4 male and 4 female Itga8-CreER^T2 ;Cav1.2 ^f/f mice are summarized in Figure 4. Vessels from both male and female mice were evaluated separately. Two-way ANOVAs with repeated measures were used to assess possible differences in contractile parameters (normalized amplitude, frequency, ejection fraction, tone, FPF and passive diameter) of popliteal lymphatics from Cav1.2^f/f mice. No significant differences were found between the parameters of male and female vessels. Likewise, two-way ANOVAs with repeated measures were used to assess possible differences in contractile parameters of popliteal lymphatics from Itga8-CreER^T2 ;Cav1.2 ^f/f mice. No significant differences were found between the parameters of male and female vessels. However, there were significant impairments in all contraction parameters, except tone, at every pressure in vessels from Itga8-CreER^T2 ;Cav1.2 ^f/f mice compared to Cav1.2^f/f mice. The normalized contraction amplitudes of Cav1.2 ^f/f control vessels were consistent with those reported in our previous studies (Castorena-Gonzalez et al., 2018a; To et al., 2018; Zawieja et al., 2018), with a peak AMP at pressures of 1-2 cm H₂O, a slight reduction at 0.5 cm H₂O and progressive decline at pressures >2 cmH₂O (Figure 4A). In contrast, normalized contraction amplitudes of Cav1.2-deficient vessels were almost negligible at all pressures. The calculated EF of control vessels exceeded 0.8 at low pressures and declined to 0.35 as pressure was elevated to 10 cm H₂O. EF of Cav1.2-deficient vessels was <0.1 at all pressures (Figure 4B). The contraction frequency of control vessels increased from ∼3 min⁻¹ to ∼16 min⁻¹ over the pressure range 0.5–10 cm H₂O, in contrast to Cav1.2-deficient vessels, in which threshold contractions were rare (Figure 4C). 5 of 8 male vessels and 6 of 9 female vessels from Itga8-CreER^T2 ;Cav1.2 ^f/f mice had no contractions larger than 4 μm at any pressure such that the average FREQ was <2 min⁻¹ at all pressures. Calculations of FPF, from EF and FREQ, predicted that Cav1.2-deficient vessels were incapable of actively transporting lymph over the entire pressure range (Figure 4D). Interestingly, the tone of Cav1.2-deficient lymphatic vessels was 50–75% higher than that of control vessels at most pressures, although none of differences were statistically significant (Figure 4E). The same pattern was observed previously in Myh11-CreER^T2 ;Cav1.2 ^f/f vessels (To et al., 2018), although tone in that study was even higher in Cav1.2-deficient vessels (reaching ∼30%) and was significantly different at most pressures. The passive diameters of Itga8-CreER^T2 ;Cav1.2 ^f/f popliteal lymphatics were not significantly different than those of control popliteal lymphatics, with a single exception for male mice at one pressure (Figure 4F).

Examples of pump tests in control and Cav1.2-deficient vessels are shown in Figures 5A, B. In the control vessel, the valve trace indicates that the outflow valve was open (position = 1) most of the time during the lymphatic contraction cycle (prior to the start of the Pout ramp). This agrees with our previous finding that the valves have an open bias in the absence of a trans-valve pressure difference. The valve closed (position = 0) transiently near the end of diastole in each contraction cycle, which we previously showed corresponds with a transient dip in internal pressure (the “suction effect”) (Jamalian et al., 2017). Shortly after the start of the Pout ramp, when Pout exceeded Pin by ∼0.3 cm H₂O, the valve switched to the closed state for the majority of the contraction cycle (up arrow, Figure 5A), except for a brief period at the peak of systolic ejection, when it transiently opened. This pattern continued to occur as Pout progressively increased, until a point was reached when the valve ‘locked’ open in diastole (down arrow, Figure 5A). The value of Pout associated with the last successful ejection (minus Pin) was an estimate of the pump limit (and probably an underestimate of the true value due to valve “lock”). In contrast, the outflow valve was open initially in a 2-valve popliteal vessel from an Itga8-CreER^T2 ;Cav1.2 ^f/f mouse, never closed transiently during the contraction cycle at equal Pin and Pout, closed when Pout exceeded Pin, and never opened until the Pout ramp was terminated (Figure 5B). This behavior is indicative of a completely ineffective active pump.

Pump limit values for 2-valve lymphangions from 3 male and 3 female Cav1.2 ^f/f mice and 4 female and 2 male Igta8-CreER^T2 ;Cav1.2 ^f/f mice are summarized in Figure 5C. The data from male and female mice were tested using a one-way ANOVA using a Kruskal-Wallis nonparametric test and Dunn’s post hoc tests. There were no significant differences between male and female vessels for either of the two genotypes, but the differences between control and Cav1.2-deficient vessels were highly significant for both male and female mice. None of the vessels from Igta8-CreER^T2 ;Cav1.2 ^f/f mice were able to generate sufficient pump strength to open an output valve at any value of Pout, confirming that active lymphatic pumping is eliminated by Cav1.2 deficiency.