Human amyloidogenic light chain proteins result in cardiac dysfunction, cell death, and early mortality in zebrafish

Abstract

Systemic amyloid light-chain (AL) amyloidosis is associated with rapidly progressive and fatal cardiomyopathy resulting from the direct cardiotoxic effects of circulating AL light chain (AL-LC) proteins and the indirect effects of AL fibril tissue infiltration. Cardiac amyloidosis is resistant to standard heart failure therapies, and, to date, there are limited treatment options for these patients. The mechanisms underlying the development of cardiac amyloidosis and AL-LC cardiotoxicity are largely unknown, and their study has been limited by the lack of a suitable in vivo model system. Here, we establish an in vivo zebrafish model of human AL-LC-induced cardiotoxicity. AL-LC isolated from AL cardiomyopathy patients or control nonamyloidogenic LC protein isolated from multiple myeloma patients (Con-LC) was directly injected into the circulation of zebrafish at 48 h postfertilization. AL-LC injection resulted in impaired cardiac function, pericardial edema, and increased cell death relative to Con-LC, culminating in compromised survival with 100% mortality within 2 wk, independent of AL fibril deposition. Prior work has implicated noncanonical p38 MAPK activation in the pathogenesis of AL-LC-induced cardiotoxicity, and p38 MAPK inhibition via SB-203580 rescued AL-LC-induced cardiac dysfunction and cell death and attenuated mortality in zebrafish. This in vivo zebrafish model of AL-LC cardiotoxicity demonstrates that antagonism of p38 MAPK within the AL-LC cardiotoxic signaling response may serve to improve cardiac function and mortality in AL cardiomyopathy. Furthermore, this in vivo model system will allow for further study of the molecular underpinnings of AL cardiotoxicity and identification of novel therapeutic strategies.

Keywords: amyloidosis; apoptosis; in vivo model cardiovascular disease; p38 mitogen-activated protein kinase.

Figures

Fig. 1.

Amyloid (AL) light chain (AL-LC) induces cardiac dysfunction in zebrafish. A: cardiac visualization under bright-field microscropy, with the inset showing an enlargement of the heart. B, left: still image frames captured during diastolic and systolic phases of cardiac contraction. Right, schematic diagrams demonstrating the cardiac dimensions used for contractile function measurements. LA, long axis; SA, short axis; d, diastole; s, systole. Cardiac contractions were captured in 72 h postfertilization embryos, and sequential images were analyzed to determine cardiac dimensions. C: stroke volume. D: heart rate. E: cardiac output. Veh, vehicle; Con-LC, control human immunoglobulin LC. n = 6 animals/group. *P < 0.05.

Fig. 2.

AL-LC induces pericardial edema in zebrafish. Cardiac injection of AL-LC resulted in fluid retention and edema in the pericardium 24 h postinjection. Left: bright-field microscopy of embryos at ×2.5 magnification injected with Veh (A), Con-LC (B), and AL-LC (C). Right: enlargement showing ×10 magnification of the cardiac region.

Fig. 3.

AL-LC induces cardiomyocyte death in zebrafish. A: active caspase 3 expression was measured using immunoblot analysis, with GAPDH as a loading control. Fifteen zebrafish were homogenized per experiment; n = 3 experiments. B: hearts were isolated from zebrafish 3 days postinjection and stained for TUNEL-labeled nuclei with 4′,6-diamidino-2-phenylindole (DAPI) counterstain. n = 6 zebrafish/group. C: cardiac-specific annexin expression was quantified 3 days after LC injection. Hearts were isolated, costained with DAPI, and imaged to determine cardiomyocyte death. n = 5 zebrafish/group. *P < 0.05.

Fig. 4.

Electron microscopy shows the absence of AL fibril infiltration. A–C: transmission electron micrographs of the cardiac chamber in Veh-injected (A), Con-LC-injected (B), and AL-LC-injected (C) zebrafish 3 days postinjection (5 days postfertilization). Left: micrographs taken at a magnification of ×690. Blood cells were visible within the cardiac chamber, and striated skeletal muscle was observed adjacent to the wall of the chamber. Right: micrographs taken at a magnification of ×6,800. Striated sarcomeric patterning of the cardiomyocytes can be observed with no detectable fibril deposition.

Fig. 5.

AL-LC injection leads to loss of survival. A: Kaplan-Meier analysis of survival after the injection of Veh, Con-LC, or AL-LC. Survival was monitored daily. n ≥ 60 zebrafish/group. B: percent survival of zebrafish 3 days postinjection with Veh, Con-LC from 2 separate patients (1 and 2), or AL-LC isolated from 3 separate patients (1–3). n ≥ 75 zebrafish/group. *P < 0.01.

Fig. 6.

AL-LC injection leads to loss of survival in a dose-dependent manner. A–C: Kaplan-Meier analysis of survival after the injection of Veh, Con-LC, or AL-LC at varying dose concentrations of 10 μg/ml (A), 100 μg/ml (B), or 1,000 μg/ml (C). Survival was monitored daily. n ≥ 75 zebrafish/group. *P < 0.01.

Fig. 7.

p38 MAPK inhibition prevents AL-LC-induced cardiac dysfunction in vivo. Color Doppler echocardiography in zebrafish embryos is shown. A: color Doppler image of an embedded zebrafish embryo. The schematic indicates the orientation of cardiac chambers and placement of the probe. B: representative color Doppler peak flow tracings of zebrafish 3 days postfertilization with and without concomitant treatment with the p38 MAPK inhibitor SB-203580 (SB). C: quantification of peak flow measurements. n = 5–6 zebrafish/group. *P < 0.05.

Fig. 8.

p38 MAPK inhibition prevents AL-LC-induced toxicity in vivo. A: Kaplan-Meier survival analysis of zebrafish treated with SB at the time of injection of Veh, AL-LC, or Con-LC. Median survival is indicated by the dotted line. B: Kaplan-Meier survival analysis of zebrafish treated with SB 5 days after the injection of Veh, AL-LC, or Con-LC. C: representative images of hearts isolated from zebrafish 72 h postinjection and stained for TUNEL-positive nuclei with DAPI counterstain. D: quantification of TUNEL-positive nuclei. n = 4–7 zebrafish/group. E: active caspase 3 expression measured using Western blot analysis. Fifteen zebrafish were homogenized per experiment; n = 2 experiments. F: phospho-p38 expression measured by Western blot analysis. Fifteen zebrafish were used per experiment; n = 2 experiments. *P < 0.05.