Animal procedures. All procedures were approved by the animal care committee at the University of Missouri and complied with the standards stated in the “Guide for the Care and Use of Laboratory Animals” (National Institutes of Health, revised 2011). The study is reported in accordance with ARRIVE guidelines.
Animals. C57BL/6J wild-type (WT) mice were purchased from Jackson Laboratory (JAX, Bar Harbor, ME, USA). Cav3.1-/- (Cacna1g null) mice on the C57BL/6J background, originally generated by Hee-Sup Shin (Korea Institute of Science and Technology; 37, were a gift from Jeffrey Molkentin (University of Cincinnati), and rederived at MMRRHC, Columbia, MO, in the C57Bl/6 background. Cav3.2-/- mice, originally generated by Chen et al. 38, were obtained from JAX (B6;129-Cacna1h,tm1Kcam./J; #013770), bred into the C57Bl/6 background for at least 8 generations. Cav3.2-/- and Cav3.1-/- mice were bred to generate Cav3.1/-;Cav3.2-/- double KO mice on the C57Bl/6 background. Myh11-CreERT2 mice (B6.FVB-Tg(Myh11-cre/ERT2)1Soff/J), obtained from Dr. Stefan Offermanns, were bred with Cav1.2f/f mice (Cacna1ctm3Hfm/J; #024714), which were purchased from JAX, to generate Myh11-CreERT2;Cav1.2l/l mice (hereafter referred to as Cav1.2 smKO mice). All genotypes were verified by PCR. Mice from the latter strain were injected with tamoxifen (10mg/ml, 100ml i.p.) for 5 days and allowed to recover for 2 weeks before being used for experiments. Mice were provided ad libitum access to food and water and housed under normal light and dark cycles in cages of up to five mice. Mice of either sex (except for Cav1.2 smKO mice) were studied at 5–10 weeks of age (18–25 g).
Lymphatic vessel isolation. Mice were anesthetized with pentobarbital sodium (60mg kg− 1, i.p.). An incision was made on the dorsal-medial side of either leg from the ankle to the groin to access the popliteal lymphatics. An excised lymphatic vessel was pinned on a Sylgard platform (Sylgard® 184, Dow Corning, Midland, MI, USA) in Krebs’ buffer supplemented with 0.5% albumin, and isolated by dissection from the surrounding connective tissue and fat. After surgery, the animal was euthanized.
Pressure myography. An excised lymphatic vessel containing at least one valve was transferred to a 3 mL chamber where it was cannulated onto two micropipettes and pressurized. The bath was exchanged at a rate of 0.5 ml/min with Krebs buffer and equilibrated for 30–60 minutes at 37oC with pressure set to 3 cmH2O, as previously described 14. The pipettes contained 0.5% albumin-supplemented Krebs buffer. Vessels used for further experimentation (except those from Cav1.2 smKO mice) developed robust, spontaneous contractions, with contractions that were entrained over the entire vessel length and amplitudes exceeding 30% at pressure = 3 cmH2O. Inner diameter at a representative region was measured continuously from video images using digital edge-detection 39. Pressures and diameter were digitized using a National Instruments A-D system (Austin, TX) under the control of a LabVIEW program as described previously 40.
Sharp electrode recordings of Vm. In separate experiments, Vm was recorded in the smooth muscle cell layer of pressurized WT mouse lymphatic vessels to verify the extent of PIN-induced hyperpolarization after L-type VGCC inhibition. To permit stable recordings of Vm in contracting vessels, wortmannin (1–3 mM, 20–30 min) was used to inhibit myosin light chain kinase and blunt vessel movement; the concentration and exposure time were adjusted to preserve minimal contractions (< 5 microns) that confirmed preservation of viability. The smooth muscle layer was impaled with an intracellular microelectrode (300–350 MW) filled with 1M KCl, and Vm was recorded using a NPI SEC-05x amplifier (ALA instruments, Farmingdale, NY) as previously described 31. The amplifier output was digitized at sampled at 1 KHz using a D-A interface (National Instruments). After a successful impalement, Vm was allowed to stabilize for 15–30 seconds. The most negative value during the AP was approximately − 35 mV. After recording multiple contraction cycles, 1 mM NIF was added to the bath solution to inhibit L-type Ca2+ channels. In some cases the impalement was lost due to the mixing procedure and, when that happened, attempts were made to impale the same cell or an adjacent cell and continue the protocol. Subsequently, PIN was added in cumulative concentrations (0.3, 1, 3 mM) while recording Vm. Once the recording was completed, the electrode was retracted from the cell and the recorded values were corrected for the offset potential.
Electric field stimulation. EFS was achieved using two 0.5 mm platinum wires (Warner Instruments, #64-1942), separated by 2.5 mm within the 3 mL bath chamber. The wires were positioned 2 mm above the bottom of the observation chamber and insulated except for the terminal 4 mm. The cannulated vessel was positioned 1 mm from the chamber bottom, equidistant between the two wires. A Grass S48 stimulator provided the depolarizing current. Initial tests showed that single twitch contractions, of amplitude comparable to those of spontaneous contractions, could be elicited with short duration (< 1 mS), single pulses of 80-90V. 90V pulses were routinely used to ensure consistent responses. The synch output of the stimulator was amplified and digitized using an A-D interface (National Instr., Austin TX) to document pulse delivery in register with the diameter recording. For EFS protocols, pressure was usually set to either 1 or 2 cmH2O, depending on the spontaneous contraction rate, to provide a contraction pattern with a sufficiently long diastolic period to allow for single EFS pulses to be delivered in lymphatic diastole.
Contraction wave analysis. To quantify the degree of entrainment of EFS-evoked contraction waves, brightfield videos of spontaneous contractions were acquired at video rates ranging from 30 to 50 fps. Recorded videos were then stored for offline processing, analysis, and quantification of the conduction speed. Videos of contractions were processed frame by frame to generate two-dimensional spatiotemporal maps (STMs) representing the measurement of the outside diameter (encoded in 8-bit grayscale) over time (horizontal axis) at every position along the vessel (vertical axis), as described previously 3. All video processing and analyses were performed using a set of custom-written Python programs. Conduction speed was determined for each wave by the slope of the corresponding band on the ST map (by linear fit of the points defining the leading edge) and the speeds were averaged for all the contractions in a given video.
Experimental Protocols. After a vessel established a consistent pattern of spontaneous contractions, one of two protocols was conducted.
The first protocol assessed the concentration-dependent inhibition by NIF on spontaneous contractions. After equilibration and establishment of a consistent pattern of spontaneous contractions at constant pressure, bath perfusion was stopped and NIF was added in cumulative concentrations (1 nM to 10 µM) to the bath. Pressure was set at either 1 or 2 cmH2O, depending on the spontaneous contraction rate of a given vessel. Contraction responses were recorded for 2–3 min before the next concentration was applied and the protocol was completed within 20 min, a time period found previously not to produce significant effects on contraction FREQ or AMP due to bath evaporation.
For the second protocol, single voltage pulses (typically 0.1–0.3 mS, 90 V) were applied during the diastolic phase of the contraction cycle, with the pulses delivered 30–60 sec apart and timed to produce minimal disruption to the spontaneous contraction pattern; this was repeated 3 times. With pressure maintained at 3 cmH2O, the bath perfusion was stopped and TTX (1 µM) applied. After assessing the effect of TTX on the contraction pattern for 3–4 min, three identical stimulus pulses were again delivered (30–60 sec apart). For WT vessels, NIF (1 µM) was subsequently added to the bath and after 4 min the stimulus pulses were repeated. In a similar set of tests, vessels from Cav1.2 smKO mice were used in lieu of NIF treatment. In both cases the KATP channel activator, pinacidil (PIN), was then added to the bath in increasing concentrations (0.3, 1, 3 µM) to hyperpolarize LMCs, allowing 2–3 min equilibration at each concentration before delivering stimulus pulses. Each time a drug was added to the bath the light path was temporarily blocked to create a vertical blanking artifact on the diameter trace. Tests using the same protocol were conducted on vessels from Cav3.1-/-;Cav3.2-/- mice. In each case the total protocol was completed in less than 20 min.
At the end of either protocol, the vessel was equilibrated for 30 min in Ca2+-free Krebs buffer solution containing 3 mM EGTA and the passive diameters at 1, 2 and 3 cmH2O were measured. Once an experiment was complete, internal diameter traces of spontaneous contractions were analyzed off-line using custom-written LabVIEW programs to detect end diastolic diameter (EDD), end systolic diameter (ESD), and contraction frequency (FREQ), each computed on a contraction-by-contraction basis and averaged over a 2–5 min period. In some cases where diameter tracking was noisy or inaccurate, the diameter was retracked during replay of 30-fps bright-field videos taken during the experiment. The diameter data were used to calculate commonly reported parameters that characterize the contractile function of lymphatic vessels:
$$\text{Amplitude (AMP)= EDD-ESD}$$
1
$$\text{Normalized AMP= }\left(\frac{\text{EDD-ESD}}{{\text{D}}_{\text{MAX}}}\right)\text{×100}$$
2
$$\text{Ejection Fraction (EF)= }\left[\frac{{\text{EDD}}^{\text{2}}\text{-}{\text{ESD}}^{\text{2}}}{{\text{EDD}}^{\text{2}}}\right]$$
3
$$\text{Fractional Pump Flow (FPF)= EF∙FREQ}$$
4
where DMAX represents the maximum passive diameter (obtained after incubation with calcium-free Krebs solution) at the intraluminal pressure used in the protocols.
Solutions and chemicals. Krebs buffer contained (in mM): NaCl, 146.9; KCl, 4.7; CaCl2, 2; MgSO4, 1.2; NaH2PO4.H2O, 1.2; NaHCO3, 3; Na-HEPES, 1.5; D-glucose, 5 (pH 7.4, 37oC), equilibrated with room air. All chemicals were obtained from Sigma (St. Louis, MO, USA), except for BSA (US Biochemicals; Cleveland, OH, USA), MgSO4, HEPES (Fisher Scientific; Pittsburgh, PA, USA), tetrodotoxin (TTX, Alomone, Israel). Nifedipine (NIF) and pinacidil (PIN) were dissolved in DMSO at stock concentrations of 1 or 10 mM. Tetrodotoxin (TTX) was dissolved in citrate buffer (1 mM).
Data analysis. Data were collected and analyzed using LabVIEW (National Instruments, Austin TX), Excel (Microsoft, Redmond, WA) and Prism 8 (Graphpad, La Jolla, CA, USA). Original recordings were plotted in IGOR (Wavemetrics, Oswego, OR). IC50 values were determined in Prism or IGOR. The four standard tests in Prism for normality (Anderson-Darling, D’Agostino & Pearson, Shapiro-Wilk, Kolmogorov-Smirnov) were used to evaluate each data set and revealed that at least half of the data sets were not normally distributed. Subsequently, one-way ANOVAs with Krusal-Wallis post-hoc tests were performed to compare the amplitude of spontaneous and EFS-induced contractions across pharmacological treatments for each genotype, and Wilcoxon matched pairs signed rank tests were used to compare pairs of data sets within each genotype. The specific tests used for each protocol are indicated in the figure legends. The data are expressed as mean ± standard error of the mean. P values < 0.05 were considered statistically significant, but other significance levels are marked when appropriate. N refers to the number of animals and n refers to the number of vessels or cells included per group.