A Caenorhabditis elegans-based assay recognizes immunoglobulin light chains causing heart amyloidosis

Abstract

Poor prognosis and limited therapeutic options characterize immunoglobulin light-chain (AL) amyloidosis with major heart involvement. Reliable experimental models are needed to study light-chain (LC)/heart interactions and to explore strategies for prevention of cardiac damage. We have exploited the nematode Caenorhabditis elegans as a novel tool, because its pharynx is evolutionarily related to the vertebrate heart. Our data demonstrate that the pharyngeal pumping of C elegans is significantly and selectively reduced by LCs from AL patients suffering from cardiomyopathy, but not by amyloid LCs with different organ tropism or nonamyloidogenic LCs from multiple myeloma. This functional alteration is dependent on the LC concentration and results in persistent pharyngeal dysfunction and in a significant reduction of the worms' lifespan. These manifestations are paralleled by an increase of mitochondrial reactive oxygen species and can be prevented by treatment with antioxidant agents. In conclusion, these data indicate that this nematode-based assay is a promising surrogate model for investigating the heart-specific toxicity of amyloidogenic LCs and for a rapid screening of new therapeutic strategies.

© 2014 by The American Society of Hematology.

Figures

Figure 1

Characterization of the effects of selected LC on the pumping rate. (A) Time-dependent effect of the amyloidogenic cardiotoxic protein (H6-BJ) and the nonamyloidogenic one (MM2-BJ) on the pumping rate of worms. Proteins, in 10 mM PBS (pH 7.4), were administered to worms at 100 µg/mL. Control worms received vehicle alone (Vehicle). Nematodes (100 worms/100 µL) were incubated with LCs for 2 hours in the absence of OP50 E coli and then plated on NGM plates seeded with bacteria. The pharyngeal pumping was scored at different times after plating (2-48 hours). Data are expressed as the mean ± standard error (SE) (n = 20 worms/group). *P < .01 vs vehicle and MM2-BJ, Student t test. (B) Dose-response effect of 1 to 200 µg/mL of H6-BJ and MM2-BJ. Mean ± SE (n = 40 worms/group). *P < .01 vs MM2-BJ, Student t test. (C) Effect of H6-BJ on the feeding behavior. Feeding assay was performed by monitoring the ability of worms to ingest multifluorescent beads. H6-BJ protein (100 µg/mL) in 10 mM PBS (pH 7.4) or vehicle alone (Vehicle) were administered to worms. Representative images, obtained from the overlay of a contrast phase and epifluorescence, indicated the presence of fluorescent beads (black arrows) in the pharynx of control worms, but not in those fed H6-BJ protein. (D) Dose-response effect of 1 to 200 µg/mL of recombinant cardiotoxic (H3-r) or noncardiotoxic (K3-r) proteins. Mean ± SE (n = 30 worms/group). *P < .01 vs K3-r, Student t test. (E) Comparison of the dose-response curves obtained for H3-r and H6-BJ proteins. IC50 values ± standard deviation were reported. The 2 proteins, at 100 µg/mL, similarly inhibited the pumping rate of worms (from 235.0 ± 3.6 pumps/min of vehicle to 177.6 ± 3.2 pumps/min and 170.4 ± 2.8 pumps/min for H6-BJ and H3-r, respectively).

Figure 2

Effect on the pumping rate of LC with different organ tropism, purified from different patients’ urine and serum or obtained as recombinant. Effect of (A) BJ LC (100 µg/mL) purified from 8 patients (3 heart AL, 1 kidney AL, 1 soft tissue AL, 3 MM), (B) serum-free LC (50 µg/mL) purified from 11 patients (5 heart AL, 4 kidney AL, 2 MM), and (C) recombinant LC (100 µg/mL) obtained from 4 patients (3 heart AL and 1 kidney AL) on pharyngeal pumping. Nematodes (100 worms/100 µL) were incubated for 2 hours, in the absence of OP50 E coli, with different amyloidogenic noncardiotoxic or cardiotoxic LCs or nonamyloidogenic LCs from patients with MM. The pharyngeal pumping rate was scored 20 hours after plating the worms on NGM agar plates seeded with fresh OP50 E coli as food. Control worms received vehicle alone (Vehicle). Each dot on the scatterplot represents the mean value of pumps per minute obtained for each single protein from 3 independent assays (n = 30 worms/assay). These values were used to calculate the mean ± SE for LCs with the similar organ tropism (horizontal line) and to perform a statistical comparison across the different groups. **P < .001 vs vehicle, °P < .05 and °°P < .01 noncardiotoxic vs cardiotoxic LCs, according to 1-way ANOVA followed by Bonferroni post hoc test.

Figure 3

Cardiotoxic LC affects nematode survival and pharyngeal ROS generation. (A) Kaplan-Meier survival curves of worms treated with vehicle, 100 µg/mL of amyloidogenic BJ cardiotoxic protein (H6-BJ), or 100 µg/ml of nonamyloidogenic BJ protein (MM2-BJ). Survival is expressed as a percentage of the initial population (3 independent experiments, n = 30 worms/group). Median survival was 14 days for vehicle, 9 days for H6-BJ (P = .024 vs vehicle, log-rank test), and 14 days for MM2-BJ (P = .072 vs worms treated with H6-BJ and P = .53 vs vehicle, log-rank test). (B) Representative images obtained from the overlay of a contrast phase and MitoSOX fluorescence. N2 worms (at L3-L4 larval stage) were fed 2 hours with vehicle or 100 µg/mL of MM2-BJ or H6-BJ. Positive control worms were fed 0.1 mM hydrogen peroxide (H2O2) for 30 min. Nematodes were then transferred to NGM plates seeded with fresh OP50 E coli and 10 µM MitoSOX Red dye. Original magnification ×40 was used. Arrows indicate the radical superoxide generation in the mitochondria of pharyngeal bulb of C elegans fed cardiotoxic H6-BJ protein or H2O2.

Figure 4

Protective effect of antioxidants, TETRA, and EGCG on the pharyngeal dysfunction caused by cardiotoxic LC. Dose-dependent effect of (A) NAC, (B) ascorbic acid, (C) TETRA, or (D) EGCG. Worms were fed for 2 hours with 100 µg/mL amyloidogenic cardiotoxic protein (H6-BJ) in the absence or presence of increasing concentrations of drugs. Control worms were fed with vehicle alone (dotted line). The pharyngeal pumping rate was scored 20 hours after plating on NGM plates, as described in “Methods.” IC50 value was calculated for each compound. (E) The effect of 5 mM NAC, 284 µM ascorbic acid, 50 µM TETRA, or 100 µM EGCG was determined by incubating worms for 2 hours with 100 µg/mL H6-BJ. The effect of the administration of each compound, alone, at the same concentration and H6-BJ alone was also evaluated. The pharyngeal pumping rate was scored 20 hours after plating on NGM plates, as described in “Methods.” Control worms were fed vehicle alone (vehicle). Mean ± SE (n = 30). **P < .01 and *P < .05 vs vehicle, °°P < .01 vs H6-BJ alone according to 1-way ANOVA followed by Bonferroni post hoc test.