Cardiac Light Chain Amyloidosis: The Role of Metal Ions in Oxidative Stress and Mitochondrial Damage

Abstract

Aims: The knowledge of the mechanism underlying the cardiac damage in immunoglobulin light chain (LC) amyloidosis (AL) is essential to develop novel therapies and improve patients' outcome. Although an active role of reactive oxygen species (ROS) in LC-induced cardiotoxicity has already been envisaged, the actual mechanisms behind their generation remain elusive. This study was aimed at further dissecting the action of ROS generated by cardiotoxic LC in vivo and investigating whether transition metal ions are involved in this process. In the absence of reliable vertebrate model of AL, we used the nematode Caenorhabditis elegans, whose pharynx is an "ancestral heart."

Results: LC purified from patients with severe cardiac involvement intrinsically generated high levels of ROS and when administered to C. elegans induced ROS production, activation of the DAF-16/forkhead transcription factor (FOXO) pathway, and expression of proteins involved in stress resistance and survival. Profound functional and structural ROS-mediated mitochondrial damage, similar to that observed in amyloid-affected hearts from AL patients, was observed. All these effects were entirely dependent on the presence of metal ions since addition of metal chelator or metal-binding 8-hydroxyquinoline compounds (chelex, PBT2, and clioquinol) permanently blocked the ROS production and prevented the cardiotoxic effects of amyloid LC. Innovation and Conclusion: Our findings identify the key role of metal ions in driving the ROS-mediated toxic effects of LC. This is a novel conceptual advance that paves the way for new pharmacological strategies aimed at not only counteracting but also totally inhibiting the vicious cycle of redox damage. Antioxid. Redox Signal. 27, 567-582.

Keywords: amyloid; caenorhabditis elegans; immunoglobulin light chain; metals; mitochondria; protein misfolding.

FIG. 1.

Effect of metal ions on the ability of LC to generate ROS and affect pharyngeal pumping in worms. (A) H₂O₂ produced by cardiotoxic LC (H7-BJ) and myeloma (MM2-BJ) incubated 2 h with or without 5 mg/ml chelex. Mean ± SE of fluorescence intensity (FI), n = 6, **p < 0.01 vs. cardiotoxic LC and °°p < 0.01 vs. myeloma, one-way ANOVA and Bonferroni's post hoc test. (B) Worms were fed for 2 h with 100 μg/ml cardiotoxic LC (H6-BJ, H7-BJ, H7-r, H18-BJ), myeloma proteins (MM2-BJ, MM4-BJ, MM7-BJ) with or without 5 mg/ml chelex or 10 μM ethylenediaminetetraacetic acid. Control worms were incubated with 10 mM PBS, pH 7.4 (Vehicle) only (dotted line). The mean ± 95% CI of pumps/min was calculated (horizontal line). Each dot is the mean of pumps/min for each protein (3 independent assays, n = 30 worms/assay). **p < 0.01 vs. Vehicle, °°p < 0.01 vs. cardiotoxic LC, one-way ANOVA and Bonferroni's post hoc test. (C) H₂O₂ produced by 45 μM cardiotoxic LC and myeloma treated with chelex and incubated 2 h with 50 μM CuCl₂, ZnCl₂, or FeCl₂. Control samples were incubated with chelex-treated 10 mM PBS, pH 7.4. Mean ± SE of FI, n = 12, **p < 0.01 vs. cardiotoxic LC incubated with chelex-treated PBS, pH 7.4, °°p < 0.01 vs. myeloma LC + chelex-treated PBS, pH 7.4, one-way ANOVA and Bonferroni's post hoc test. (D) Worms were fed for 2 h with 100 μg/ml cardiotoxic LC (H7-BJ), myeloma (MM2-BJ) with or without 50 μM CuCl₂, ZnCl₂, or FeCl₂. Control worms were incubated with 10 mM PBS, pH 7.4 (Vehicle) only or 50 μM CuCl₂, ZnCl₂, and FeCl₂. Pumping rate as mean pumps/min ± SE (n = 20 worms/assay, three assays). **p < 0.01 versus vehicle, one-way ANOVA and Bonferroni's post hoc test.°°p < 0.0001 versus cardiotoxic LC, two-way ANOVA and Bonferroni's post hoc test. (E) H₂O₂ produced by cardiotoxic LC (H7-BJ) and myeloma (MM2-BJ) previously treated for 3 h at 20°C with iodoacetamide and then incubated 2 h with 50 μM CuCl₂. Mean ± SE of FI, n = 9, **p < 0.01 versus cardiotoxic LC not treated with iodoacetamide and °°p < 0.01 vs cardiotoxic LC treated with iodoacetamide, one-way ANOVA and Bonferroni's post hoc test. ANOVA, analysis of variance; BJ, Bence Jones; LC, immunoglobulin light chain; PBS, phosphate-buffered saline; ROS, reactive oxygen species.

FIG. 2.

Effect of metal-binding compounds CQ and PBT2 on LC-induced H₂O₂ production and oxidative damage. (A) Dose–response effect of CQ and PBT2 on LC-induced pharyngeal dysfunction. Worms were fed for 2 h with 100 μg/ml cardiotoxic LC in the absence or presence of 0–25 μM CQ or 0–25 nM PBT2. Control worms received vehicle alone (dotted line). Each value is the mean ± SE, n = 30. IC₅₀ ± SD are reported, p < 0.01, Student's t-test. (B) H₂O₂ produced by cardiotoxic LC (H7-BJ) incubated 2 h with or without 2 nM PBT2 or 25 μM CQ. Mean ± SE of FI, n = 6, **p < 0.01 versus cardiotoxic LC, one-way ANOVA and Bonferroni's post hoc test. (C, D) Worms were fed for 2 h with 100 μg/ml cardiotoxic LC (H6-BJ, H7-BJ, H7-r, H18-BJ) with or without 25 μM CQ or 2 nM PBT2. H₂O₂ (1 mM) was administered for 30 min with or without the drugs. Control worms received vehicle alone (dotted line). (C) The mean ± 95% CI of pumps/min was calculated (horizontal line). Each dot is the mean of pumps/min for each protein (3 independent assays, n = 30 worms/assay). **p < 0.01 versus Vehicle, °°p < 0.01 versus cardiotoxic LC, one-way ANOVA and Bonferroni's post hoc test. (D) Images obtained from the overlay of a contrast phase and MitoSOX fluorescence (arrows). Scale bar 50 μm. (E) Kaplan–Meier survival curves, n = 30 worms/group, three independent experiments. CQ, 5-chloro-7-iodo-quinolin-8-ol.

FIG. 3.

ROS-induced cardiotoxic LC severely disrupt Caenorhabditis elegans pharyngeal ultrastructure. (A–G) Representative images of worm's pharynx obtained from the ultrastructural analysis by TEM in C. elegans fed for 2 h with (A) Vehicle, (B) myeloma protein (MM2-BJ), (C) cardiotoxic LC (H7-BJ) alone, or with (D) 25 μM CQ, (E) 2 nM PBT2, (F) 50 μM TETRA, or (G) 5 mM NAC. Images showed two pharyngeal muscles (pm) separated by a marginal cell (mc), placed at the corner of the pharyngeal channel (ch). Mitochondria are indicated by arrows. Scale bar, 500 nm. Pharyngeal muscles of worms fed cardiotoxic LC resulted in damage to mitochondria, which exhibited a clustering pattern, irregular shape, swelling, and massive disruption of the internal components (i.e., cristae). Marginal cells, which contain many mitochondria due to their active role in contractile motor function, were seriously compromised and myofilaments connected to the marginal cells, which were perfectly aligned in vehicle-fed worms, were deranged. NAC, N-acetyl-cysteine; TEM, transmission electron microscopy; TETRA, tetracycline hydrochloride.

FIG. 4.

Mitochondrial damage in heart muscle tissue of cardiac AL patients. Ultrastructural details from representative TEM images of endomyocardial biopsies from (A–C) severe cardiac amyloid AL patients and (D) a patient affected by dilatative cardiomyopathy. Although myocardial fibers (mf) are relatively well preserved in AL patients, most mitochondria (white arrows) show remarkable alterations with enlarged size and disruption (A) or total loss of cristae (B, C). LC were identified by postembedding immunogold staining with 15 nm gold-conjugated protein A (black arrows) in the interstitium and along the basement membrane of a myocardial fiber. (D) The myocardium of a patient with nonamyloid cardiomyopathy shows well-preserved mitochondria (white arrows) and glycogen deposits (g). To better highlight the differences in damage, individual mitochondria in the red insets are shown in Supplementary Fig. S7. Uranyl acetate, lead citrate. Scale bar, 1 μm.

FIG. 5.

Metal ions drive the ability of cardiotoxic LC to promote DAF-16 translocation from cytoplasm to nucleus in TJ356 transgenic worms. (A, B) Image of DAF-16::GFP expression in (A) control vehicle-fed and (B) cardiotoxic LC-fed worms (100 μg/ml H7-BJ for 2 h). (C, D) The subcellular distribution of DAF-16 expression in worms fed 2 h: vehicle, 100 μg/ml cardiotoxic LC with or without 25 μM CQ, 2 nM PBT2, 50 μM TETRA, or 5 mM NAC. According to DAF-16 localization, worms were divided into two phenotypes, including “cytosolic” and “nuclear.” The percentage of DAF-16 localization in respect to vehicle-fed worms was calculated based on three experiments, n = 100. Mean ± SE. ** p < 0.01 versus vehicle, °p < 0.05 and °°p < 0.01 versus cardiotoxic LC, one-way ANOVA and Bonferroni's post hoc test. GFP, green fluorescent protein.

FIG. 6.

Metal ions drive the ability of cardiotoxic LC to induce the pharyngeal expression of HSP-16.2 and SOD-3. (A–D) Transgenic worms were fed for 2 h with Vehicle (i, 10 mM PBS, pH 7.4), 100 μg/ml myeloma (ii, MM2-BJ), 100 μg/ml cardiotoxic LC (iii, H7-BJ), cardiotoxic LC +25 μM CQ (iv) or 2 nM PBT2 (v, cardiotoxic LC + PBT2). (A) Images of HSP-16.2 expression as overlays of GFP fluorescence and light microscopy in CL2070 transgenic worms. Scale bar, 50 μm. (C) Images of SOD-3 expression as GFP fluorescence (arrows) in CF1553 transgenic worms. Scale bar, 50 μm. Quantified GFP intensity in (B) CL2070 and (D) CF1553 worms in response to treatments. FI in each group was calculated as mean gray value ± SE based on three experiments, n = 25. **p < 0.01 versus Vehicle, °p < 0.05 and °°p < 0.01 versus cardiotoxic LC, one-way ANOVA and Bonferroni's post hoc test. HSP, heat shock protein; SOD, superoxide dismutase; FI, fluorescence intensity.

FIG. 7.

Synergistic beneficial effect of PBT2 and TETRA. (A, B) Pharyngeal performance of worms fed 100 μg/ml cardiotoxic LC (H7-BJ) for 2 h and then treated for 30 min with (A) 25 μM CQ, 20 μM TETRA, or 2 nM PBT2 or with (B) 0.5–2 nM PBT2 alone or together with 20 μM TETRA. Control worms fed vehicle alone. **p < 0.001, *p < 0.005 versus vehicle, °°p < 0.001 versus cardiotoxic LC, one-way ANOVA and Bonferroni's post hoc test. ^§§p < 0.01 significant interaction versus worms fed cardiotoxic LC +0.5 nM PBT2, two-way ANOVA and Bonferroni's post hoc test.

FIG. 8.

Proposed model for metal ion involvement in the mechanism underlying the LC-induced toxicity. Based on our current knowledge, redox-active transition metals, particularly copper, drive the ability of cardiotoxic LC to produce ROS in vivo. The excessive production of ROS can directly target the pharyngeal cells, damaging the organelle functions and the ultrastructure, particularly at the mitochondrial level. ROS can be also produced as a result of the mitochondrial dysfunction requiring copper and iron for the activation of the enzymes involved in the oxidative phosphorylation pathway. Intracellular signaling events aimed at limiting and repairing the stress-induced damage are activated. Mitochondria reacts to ROS by inducing the expression of the scavenger protein SOD-3. In addition, the chaperone HSP-16.2, an αB-crystallin-related protein, is activated as well as the nuclear translocation of the FOXO/DAF-16 transcription factor. This last one can trigger a secondary ROS-induced cellular response by inducing the transcription of stress-responsive genes, including hsp-16.2 and sod-3, and controlling longevity. FOXO, forkhead transcription factors.