Independent and interactive effects of preload and afterload on the pump function of the isolated lymphangion

Abstract

We tested the responses of single, isolated lymphangions to selective changes in preload and the effects of changing preload on the response to an imposed afterload. The methods used were similar to those described in our companion paper. Step-wise increases in input pressure (P(in); preload) over a pressure range between 0.5 and 3 cmH(2)O, at constant output pressure (P(out)), led to increases in end-diastolic diameter, decreases in end-systolic diameter, and increases in stroke volume. From a baseline of 1 cmH(2)O, P(in) elevation by 2-7 cmH(2)O consistently produced an immediate fall in stroke volume that subsequently recovered over a time course of 2-3 min. Surprisingly, this adaptation was associated with an increase in the slope of the end-systolic pressure-volume relationship, indicative of an increase in contractility. Lymphangions subjected to P(out) levels exceeding their initial ejection limit would often accommodate by increasing diastolic filling to strengthen contraction sufficiently to match P(out). The lymphangion adaptation to various pressure combinations (P(in) ramps with low or high levels of P(out), P(out) ramps at low or intermediate levels of P(in), and combined P(in) + P(out) ramps) were analyzed using pressure-volume data to calculate stroke work. Under relatively low imposed loads, stroke work was maximal at low preloads (P(in) ∼2 cmH(2)O), whereas at more elevated afterloads, the optimal preload for maximal work displayed a broad plateau over a P(in) range of 5-11 cmH(2)O. These results provide new insights into the normal operation of the lymphatic pump, its comparison with the cardiac pump, and its potential capacity to adapt to increased loads during edemagenic and/or gravitational stress.

Fig. 1.

Response of an isolated lymphangion to short-lasting, step-wise elevations in input pressure (P_in). A: recording showing spontaneous lymphangion contractions at six different levels of preload (P_in), starting from 0.5 cmH₂O, with output pressure (P_out) held constant. P_in was elevated in sequential steps (no return to baseline) and maintained for ∼5–10 contractions (cf. Fig. 2). The schematic diagram of the preparation shown in the inset shows where pressures and diameter were measured. The blue trace shows P_in (in cmH₂O), the red trace shows P_out (in cmH₂O), and the black trace shows intraluminal pressure (P_L; in cmH₂O), measured by the servo-null micropipette. End-diastolic diameter (EDD) was measured at the end of diastole, and end-systolic diameter (ESD) was measured at the peak of systole. B: pressure-volume (P-V) plots of data from A, showing three complete contractions at each P_in level; each set of contractions is color coded. Volume was calculated as described in methods. Stroke volume (SV) was maximal at P_in = 3 cmH₂O in this vessel. Points a–d were used to quantify the loops, with point a showing the end of diastole, point b showing peak systolic pressure, point c showing the end of systole, and point d showing the minimal pressure during the contraction cycle. C: summary P-V diagram constructed from measurements of points a–d for 8 vessels. The solid and dotted black lines in B and C approximate the end-systolic pressure-volume relationship (ESPVR) and end-diastolic pressure-volume relationship (EDPVR), respectively. EDVPR was curvilinear. ESPVR was curvilinear for the lower range of P_in levels but linear over the higher range of P_in levels.

Fig. 2.

Time course of responses to sustained, step-wise P_in elevation. A: recording showing the pressure and diameter responses that occurred in response to a sustained P_in step. Initially, P_in was set to 0.5 cmH₂O and P_out to 1 cmH₂O (to prevent forward flow). P_in was then increased in steps to 1, 5, 8, and 12 cmH₂O for 5 min at each level, returning to the control level after each step (for this vessel, P_in was elevated using the reservoir system); P_out was held constant. Time-dependent, secondary changes in EDD, amplitude (AMP), and ESV occurred that were not evident in Fig. 1 (denoted by arrow for the first P_in step). In this particular vessel, the servo-null pipette was positioned closer to the input valve than in Fig. 1 so that P_L was closer to P_in than P_out. *Diameter tracking artifact. The third step in this recording is available as a movie (see Supplemental Movie S1, available at the American Journal of Physiology-Heart and Circulatory Physiology website). B and C: summary analysis of AMP and frequency (FREQ) changes over time for the protocol used in A plotted as a function of contraction number. Data are means ± SE. Time is relative to the P_in step. The color coding scheme is same as in Fig. 1. The open symbols indicate points that were significantly different from the point at time 0 (immediately after the respective P_in step) using ANOVA followed by Dunnett's test. The color denotations in C apply to both B and C. D: P-V plot showing selected contractions from the recording in A. The blue loops correspond to three contractions at the control P_in level (marked by the blue dot in A). Each black loop corresponds to the first contraction after the P_in step (black dot in A), and each gold loop corresponds to the contraction in which the minimal value of ESV was first reached (gold dot in A). The dotted black line approximates EDPVR, whereas the dotted lines approximate ESPVR for the initial contraction and for the contraction when a steady-state AMP was reached (ESPVR′). E: summary plot showing the average shape of P-V loops from seven vessels at various levels of P_in (afterload was held at the baseline level). Because of the unusual loop shapes in several vessels, the six standard points described in the companion paper (10) were measured for each P-V loop. The color scheme is similar to that used in Fig. 1, B and C. The open symbols represent contractions immediately after a P_in step, whereas the filled symbols represent contractions when a steady-state response had been achieved. The black and gold lines were fitted to the ESPVR (solid lines) and EDPVR (dotted lines) for the initial and steady-state time points, respectively. A modified hyperbolic function was used to fit ESPVR, whereas an exponential function was used to fit EDPVR. The steady-state curves significantly differed from the initial curves for both ESPVR (P < 0.0001) and EDPVR (P < 0.05).

Fig. 3.

Summary of lymphangion contraction parameters as a function of P_in obtained using 5-min step-wise increases in P_in with P_out held constant at 1 cmH₂O. A: AMP. B: normalized (norm) AMP. C: SV. D: EDD. E: ESD. F: tone. G: ejection fraction (EF). H: FREQ. I: fractional pump flow (FPF). Each point represents the mean ± SE. Filled black symbols represent the initial response (within 0.5 min after the pressure step), open symbols represent steady-state responses (4.5 min after the pressure step), and gray symbols indicate baseline values. n = 7 vessels except for F, where n = 4 and valid calculations of tone could not be calculated at some P_in levels for three vessels. *P < 0.05 between the 0.5- and 4.5-min time points using Bonferroni's post hoc test after two-way ANOVA.

Fig. 4.

Response to ramp-wise P_in elevation under conditions where forward flow is minimized. A: response of an isolated lymphangion to a ramp-wise increase in P_in from 2 to 16 cmH₂O with P_out held constant at 2 cmH₂O. The long pause before contractions resumed after the end of the ramp (time: 162–164 min) is consistent with rate-sensitive inhibition (8). *Brief simultaneous pulse of P_out to 12 cmH₂O to check the servo-null calibration. The dotted horizontal line shows the maximal EDD for reference (obtained at P_in = 16 cmH₂O). Valve position traces show the relative positions of the input (blue trace) and output (red trace) valve leaflets, where 1 = open and 0 = closed (for tracking details, see methods). B: response of the same lymphangion in A to a combined ramp-wise increase in P_in and P_out from 2 to 16 cmH₂O. For reference, the dotted horizontal line shows the maximal EDD (obtained at P_in = P_out ∼ 8 cmH₂O); subsequently, a slight constriction (arrow) occurred at higher levels of P_in and P_out. C: P-V relationship for the contractions during the P_in ramp in A. The blue loops correspond to three contractions at the control P_in level, and the color-coded loops correspond to the contractions shown in the corresponding colors of the trace in A. D: P-V relationship for the contractions shown in the first half of the P_in ramp in B. The blue loops indicate five contractions at the control P_in level, and the color-coded loops correspond to the corresponding colors of the trace in B. Only the contractions for the first half of the combined ramp are shown so that the ranges of P_L values are matched between C and D. The solid and dotted black lines in C and D define ESPVR and EDPVR, respectively. The slight leftward bowing of the ESPVR in C is discussed in the text.

Fig. 5.

Effect of preload on the ability of an isolated lymphangion to pump against a ramp-wise elevation in P_out. A: recording showing the response of a single lymphangion to multiple P_out ramps, each beginning at a different level of P_in. The inset shows the expanded time scale for ramp 3. With each contraction during the ramp, the P_L pulse amplitude increased to match P_out, until the ejection limit was reached (P_limit); after that point, the output valve failed to open during systole until the ramp was terminated. The output valve record is the top trace, and the gaps during the ramps denote the periods in which ejection fails. B: contraction AMP for each contraction cycle during each P_out ramp in A plotted as a single point against the instantaneous P_out level. Each curve represents the data set obtained for one P_out ramp at a given level of P_in. The family of curves is therefore analogous to a set of force-velocity curves (force vs. afterload) at different levels of preload. The color coding is similar to the scheme used in Fig. 1. C: difference between P_limit and P_in plotted as a function of P_in (filled circles) from the recording in A. The points were fit to a third-order polynomial. For comparison, the contraction AMP (averaged over at least 3 contraction cycles) before the beginning of each P_out ramp was plotted as a function of P_in (open circles; fit to a power function).

Fig. 6.

Stroke work for the various protocols calculated from the area of the P-V loops for individual contractions. A: protocol 3. B: protocol 2. C: protocol 4. D: protocol 5. See methods for protocol descriptions. A: effects of elevated P_out at two levels of P_in. B: effects of P_in at two levels of P_out. *Significantly different from control (P_in = 1 cmH₂O except in B, where P_out = 1 cmH₂O) using Dunnett's post hoc tests; ^≠significantly different from control (P_in = 3 cmH₂O in A and P_out = 3 cmH₂O in B) using Dunnett's post hoc tests.

Fig. 7.

Example of a lymphangion that exhibited preload accommodation when contraction strength was insufficient to pump against an elevated afterload. A: recordings of pressure, diameter, and valve position of an isolated lymphangion in response to a step-wise P_out increase to a relatively high value (8 cmH₂O). Control contractions at equal levels of P_in and P_out are shown in blue. After the P_out step, the output valve closed and remained closed for ∼10 contractions. Initially, the lymphangion only developed a peak systolic pressure of ∼3.5 cmH₂O. P_L pulse amplitude began to subsequently increase (to ∼6 cmH₂O), but this was insufficient to achieve ejection as the output valve continued to remain closed during systole. The input valve then closed (arrow 1), after which diastolic volume and pressure increased. At time = 104 min, ejection resumed as the resulting peak systolic pressure exceeded P_out. Subsequently, contractions became stronger, P_L pulse amplitude increased, and ejection continued as diastolic pressure returned toward control levels. At arrow 2, diastolic P_L essentially returned to control because the input valve began to open and close with each contraction cycle (indicating that P_L equalized with P_in). After P_out returned to the control level, contraction AMP (green trace) remained elevated (compared with its initial level before the P_out step), suggesting that contraction strength continued to remain elevated. B: P-V loop analysis for the first half of the trace in A. The blue loops are control contractions, corresponding to the blue portion of trace in A. The other loops correspond to the multicolored portion of the trace labeled “B,” with the time of the loops indicated by the color of the trace. The line labeled ESPVR was drawn from ESV of the control loops to ESV of the final loops (i.e., when EDP was maximal) for this portion of the trace. The lines for ESPVR′ and EDVPR are redrawn from the plot in C. C: P-V loop analysis for the second half of the trace in A. The blue loops show the control contractions for reference (corresponding to the blue portion of the trace in A). The other loops correspond to the multicolored portion of the trace in A, with the time of the loops indicated by the color of the trace. The green traces show several loops immediately after the return of P_out to its control level (corresponding to the green portion of the trace in A). The line representing EDPVR was drawn through the diastolic points for the yellow portion of the trace and then also overlaid on the curve in B for reference. The line representing ESPVR′ was drawn from the ESV for the control loops and ESV for the final loops for the yellow portion of the trace and then also overlaid on the curves in B. The curve labeled “passPVR” is the passive P-V relationship obtained with a simultaneous P_in + P_out ramp in Ca²⁺-free physiological saline solution at the end of the experiment.

Fig. 8.

Schematic diagram comparing the responses of the heart and the isolated lymphangion to changes in preload (A–C) and afterload (D–F). A: response of the cardiac ventricle to increased preload. The control cardiac cycle is loop 1 (black outline). In response to an increase in preload, the isolated heart adjusts to loop 2 (on the second and subsequent contractions, given the stated conditions). The intact heart subsequently adjusts to loop 3 on the third contraction because of a secondary increase in afterload. B: response of the lymphangion to preload elevation (P_in to P′_out),). The P-V loop initially contracts (loop 1 to loop 2) and then subsequently expands to loop 3 over the course of 5–20 contractions such that SV′ > SV. At much higher levels of preload (P″_out),), the same adaptation occurs, but the resulting SV is reduced from the control value (SV″ < SV). C: if afterload is elevated within a certain range, the P-V loop narrows (loop 1 to loop 2), but the isolated lymphangion can subsequently accommodate by increasing diastolic filling and EDP to increase contraction strength (loop 2 to loop 3). See text for discussion. D: the isolated heart responds to an increase in aortic pressure by shifting from loop 1 to loop 2, with SV decreasing in proportion to the increase in ESV. The intact heart undergoes a subsequent increase in preload (loop 2 to loop 3) due to incomplete emptying of the ventricle; over a longer time interval, the Anrep effect may also be invoked to produce a modest decrease in ESV (not shown). E: the response of the lymphangion to afterload elevation (P_out to P′_out) involves an initial narrowing of the P-V loop (loop 1 to loop 2) followed by subsequent expansion (loop 2 to loop 3) as an intrinsic increase in contractility occurs. ESPVR shifts to a higher slope. F: if afterload is elevated within a certain range, the lymphangion is initially unable to eject and the P-V loop narrows (loop 1 to loop 2); subsequently, the strength of contraction increases (loop 2 to loop 3) until ejection resumes. The results shown in E and F are based in part on data from the companion paper (10). A and D were modified from R. Klabunde (http://www.cvphysiology.com/CardiacFunction/CF025.htm).