Intrinsic increase in lymphangion muscle contractility in response to elevated afterload

Abstract

Collecting lymphatic vessels share functional and biochemical characteristics with cardiac muscle; thus, we hypothesized that the lymphatic vessel pump would exhibit behavior analogous to homeometric regulation of the cardiac pump in its adaptation to elevated afterload, i.e., an increase in contractility. Single lymphangions containing two valves were isolated from the rat mesenteric microcirculation, cannulated, and pressurized for in vitro study. Pressures at either end of the lymphangion [input pressure (P(in)), preload; output pressure (P(out)), afterload] were set by a servo controller. Intralymphangion pressure (P(L)) was measured using a servo-null micropipette while internal diameter and valve positions were monitored using video methods. The responses to step- and ramp-wise increases in P(out) (at low, constant P(in)) were determined. P(L )and diameter data recorded during single contraction cycles were used to generate pressure-volume (P-V) relationships for the subsequent analysis of lymphangion pump behavior. Ramp-wise P(out) elevation led to progressive vessel constriction, a rise in end-systolic diameter, and an increase in contraction frequency. Step-wise P(out) elevation produced initial vessel distention followed by time-dependent declines in end-systolic and end-diastolic diameters. Significantly, a 30% leftward shift in the end-systolic P-V relationship accompanied an 84% increase in dP/dt after a step increase in P(out), consistent with an increase in contractility. Calculations of stroke work from the P-V loop area revealed that robust pumps produced net positive work to expel fluid throughout the entire afterload range, whereas weaker pumps exhibited progressively more negative work as gradual afterload elevation led to pump failure. We conclude that lymphatic muscle adapts to output pressure elevation with an intrinsic increase in contractility and that this compensatory mechanism facilitates the maintenance of lymph pump output in the face of edemagenic and/or gravitational loads.

Fig. 1.

Response of an isolated lymphangion to an output pressure (P_out) ramp. A: diagram of the isolated lymphatic vessel preparation showing relative positions of the input, output, and servo-null pipettes. The red and black rectangles show the approximate positions of the diameter tracking windows used in B. B: representative recording from a lymphatic vessel responding to a ramp-wise elevation in P_out. Initially, input pressure (P_in) and P_out were both set to 1 cmH₂O, and, after several spontaneous contractions, a P_out ramp to ∼12 cmH₂O (at 4 cmH₂O/min) was imposed. The bottom trace shows the time course of the inner diameter changes in the central segment (black) and output segment (red). The pressure traces show P_in (blue), P_out (red), and intraluminal pressure (P_L; black; measured by the servo-null pipette) superimposed on a common scale. As the P_out ramp progressed, greater systolic P_L developed by the central segment during each contraction cycle, but diastolic pressure returned to 1 cmH₂O during the diastolic phase of each cycle. The top and middle traces show the input (blue) and output (red) valve positions (see methods), where 1 = open and 0 = closed. Both valves opened and closed during each contraction cycle over the entire duration of the P_out ramp, indicating that alternate phases of ejection and refilling occurred at all P_out levels shown. The lower than control contraction frequency (FREQ) after completion of the ramp is consistent with rate-sensitive inhibition (10).

Fig. 2.

Summary data describing changes in lymphangion pump parameters during P_out ramps to 16 cmH₂O. Data were binned according to 1-cmH₂O pressure intervals before analysis (as described in methods). See text for details. A: amplitude (AMP). B: end-diastolic diameter (EDD). C: FREQ. D: stroke volume (SV). E: end-systolic diameter (ESV). F: fractional pump flow (FPF). G: ejection fraction (EF). H: tone. I: calculated pump flow (CPF). *Significant difference from the control value at 1 cmH₂O.

Fig. 3.

Pressure-volume (P-V) loop analysis for the isolated lymphangion. A: time course of P_L and diameter changes during two complete contraction cycles when P_in ∼ P_out = 2 cmH₂O (left) and after P_out was elevated to ∼4 cmH₂O (right). Vertical lines denote the beginning of systole and diastole. P_in (blue) and P_out (red) traces are overlaid on the P_L trace (pressure recorded by the servo-null pipette in the central chamber) to facilitate comparisons of the different pressures at each point in time. B: plot of P_L versus calculated volume for the two contraction cycles shown in A. The color coding corresponds to the progression of time (relative to the start of contraction) from the beginning of the first contraction (dark brown) to the end (yellow) of each contraction (e.g., each color shade corresponds to the time point shown in the trace in A). The direction of both loops was counterclockwise, as indicated by arrows.

Fig. 4.

Summary analysis for P-V loops. A: response to ramp-wise P_out elevation from 1 to 16 cmH₂O followed by a return to the control P_out level. B: P-V loops plotted for the three contractions marked by the arrows in A. The color coding corresponds to the progression of time from the beginning of the first contraction (dark brown) to the end (yellow) of each contraction (e.g., each color shade corresponds to the time point shown in the trace in A). Points a–f indicate six different standard points on a typical P-V loop (see text for description). C: summary data for 16 vessels subjected to P_out steps from 1 to 4, 8, 12, or 16 cmH₂O in which measurements were made at each of the six points indicated in B. Each point represents the mean ± SE.

Fig. 5.

Stroke work at different levels of P_out. A: lymphatic vessel response to a P_out ramp from 3 to 16 cmH₂O. P_in (blue) and P_out (red) traces are overlaid on the P_L trace. The valve position trace reflects the positions of the output valve leaflets. Six representative contractions are labeled (contractions 1–6). B: time course of P_L, diameter, and calculated stroke work changes for contraction 3. C: P-V plots for contractions 1–6 in A. Red shading indicates positive stroke work (only the area inside the loop is shaded for clarity). D: plot of net stroke work as a function of P_out for all contractions in A (contractions 1–6 denoted by open circles).

Fig. 6.

Stroke work at P_out levels approaching and exceeding the limiting pressure associated with ejection (P_limit). A: lymphatic vessel response to a P_out ramp from 1 to 15 cmH₂O. P_in (blue) and P_out (red) traces are overlaid on the P_L trace. The valve position trace reflects the position of the output valve leaflets. Six representative contractions are labeled (contractions 1–6). B: time course of P_L, diameter, and calculated stroke work changes for contraction 5. C: P-V plots for contractions 1–6 in A. Red shading indicates positive stroke work, and blue shading indicates negative stroke work (only the area inside the loop is shaded for clarity). Negative stroke work coincided with contractions that failed to open the output valve. D: plot of net stroke work as a function of P_out for all contractions in A (contractions 1–6 denoted by open circles).

Fig. 7.

Secondary changes in EDV and ESV after a step elevation in P_out. A: time course of P_L and diameter changes in response to three step changes in P_out (from 1 to 12, 8, and 4, cmH₂O). Horizontal lines are reference points drawn to assess relative changes in ESD for the first P_out step; the dotted line marks the ESD for the initial contraction, and solid line marks the ESD for contractions 8–12. P_in (blue) and P_out (red) traces are overlaid on the P_L trace. The open circle denotes a spike artifact in the P_L recording from table vibration or the pipette tip touching the vessel wall. B: P-V plots for the P_out steps shown in A. The color coding corresponds to the progression of time from the beginning (dark brown) to the end (yellow) of each step. The initial one to two contractions before each pressure step are shown in dark brown (where P_out = 1 cmH₂O). The vertical lines indicate linear fits to P-V points at end systole for the first [early end-systolic pressure-volume relationship (ESPVR)] and last (late ESPVR) contractions at each P_out level. C: summary plot of average values for P_L and volume at the end of systole for the first contraction (c1), fourth contraction (c4), and eighth contraction (c8) after a P_out step, with corresponding P_out levels indicated on the right. Blue, red, and green lines are the respective best linear fits of the three sets of data points (c1, c4, and c8), and equation parameters are shown in Table 2.

Fig. 8.

Time-dependent increase in the strength of contraction after a P_out step. A: diameter and pressure recordings along with dP_L/dt analysis from a vessel responding to a 5-min P_out step from 1 to 8 cmH₂O. On the first spontaneous contraction after the step, systolic P_L rose to only 2.5 cmH₂O, suggesting that this was a relatively weak pump; the output valve failed to open. However, over the ensuing 2-min period, peak systolic P_L progressively rose until it exceeded P_out at time ∼ 114 min. Subsequently, these peak systolic pressures were maintained, and the output valve opened during each contraction cycle. The valve position trace reflects the position of the output valve leaflets. Arrow 1 indicates the first contraction associated with opening of the output valve at elevated P_out; arrow 2 indicates the beginning of a continuous series of contractions that opened the output valve. The blue trace is the control period before the P_out step used for change of diameter over time (dD/dt) analysis, and the green trace is a recovery period used for dD/dt analysis. B: P-V analysis for the selected portion of the recording marked “B” in A. The color coding corresponds to the timescale in A. The blue loops are control loops. The dotted lines show estimations of the ESPVRs for the initial five P-V loops (black) and the final five P-V loops (yellow). C: P-V analysis for the selected portion of the recording marked “C” in A, showing that the loops shifted to the left as the lymphangion successfully ejected. The color coding corresponds to the timescale in A.