Cytotoxicity of amyloidogenic immunoglobulin light chains in cell culture

Abstract

Light-chain amyloidosis (AL) is a devastating protein-misfolding disease characterized by abnormal proliferation of plasma cells in the bone marrow that secrete monoclonal immunoglobulin light chains that misfold and form amyloid fibrils, thus causing organ failure and death. Numerous reports on different protein-misfolding diseases show that soluble oligomeric species populated by amyloidogenic proteins can be quite toxic to cells. However, it is not well established whether the soluble immunoglobulin light-chain species found in circulation in patients with AL are toxic to cells in target organs. We determined the cellular toxicity of two well-characterized light-chain variable domain proteins from cardiac AL patients and their corresponding germline protein, devoid of somatic mutations. Our results show that the soluble form of the AL proteins we characterized are toxic to cardiomyocytes, and that the species found in cell culture correspond, for the most part, to the species present in circulation in these patients.

Figure 1

Cell viability in the presence of light chains followed by MTT assay. Protein concentrations from 10 to 20 μM were incubated with HL-1 cardiomyocytes for 24–72 h to determine cell viability. (a) κI O18/O8, (b) AL-09, and (c) AL-12. Samples were set up in triplicate, with average values shown from one assay

Figure 2

Percentage of living HL-1 cardiomyocytes when incubated with 12 μM soluble light-chain protein followed by MTT assay. Proteins were incubated with cells for 24–72 h. Samples were set up in triplicate in two independent assays with the average values, error bars, and P-values shown. ^*P<0.03, ^**P<0.004, ^***P<0.02

Figure 3

HL-1 cardiomyocytes caspase activity in the presence of light chains. Protein concentrations of 10–20 μM were incubated with cells for 24–72 h. Reagents were added 1 h before reading each plate. Samples were set up in triplicate with the average fluorescence shown. (a) κI O18/O8, (b) AL-09, and (c) AL-12

Figure 4

Caspase activity detected in HL-1 cardiomyocytes with a protein concentration of 12 μM. Cells were incubated with protein for 24–72 h. Reagents were added 1 h before reading plate. Average fluorescence, error bars, and P-values were calculated from triplicate samples from two independent assays. ^*P<0.008, ^**P<0.05

Figure 5

Soluble light-chain species detected in cell culture analyzed by analytical size exclusion chromatography and western blot. HL-1 cardiomyocytes were incubated with 12 μM protein for 24–72 h. Samples were set up in triplicate and combined before injecting onto the analytical size exclusion column. The molecular mass corresponding to the peaks was calculated using a standard curve described in the ‘Materials and Methods' section. (a) κI O18/O8, (b) AL-09, and (c) AL-12. The molecular weight corresponding to peaks of interest are shown. Western blot analysis shows immunoreactive κI O18/O8 (d, g, j), AL-09 (e, h, k), and AL-12 (f, i, l) species in cell culture. Molecular weights denoted in the figure are expressed in kDa